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AriaMx Real-Time PCR Software

1. Assign plate properties for a Quantitative PCR Fluorescence Probe experiment on page 88 Assign plate properties for an Allele Discrimination DNA Binding Dye experiment on page 104 Assign plate properties for an Allele Discrimination Fluorescence Probe experiment on page 111 Assign plate properties for a Comparative Quantitation experiment on page 95 Assign plate properties for a User Defined experiment on page 118 To hide the Properties panel click the arrow icon in the upper left corner of the panel Click the arrow again to display the Properties panel Agilent AriaMx Real Time PCR Program 245 12 Creating and Setting Up an MEA Project View the thermal profiles of experiments in a project Because the experiments in a MEA project are post run you cannot edit the thermal profiles However you can use the Thermal Profile screen to view the thermal profiles of the experiments In the center of the Thermal Profile screen is a visual representation of the temperature cycling program that the instrument used while running the experiment To open the Thermal Profile screen for a project Click Thermal Profile in the Experiment Area panel on the left side of the screen See Elements of a Thermal Profile on page 129 for a description of the elements of a thermal profile image To view the thermal profile for an experiment in a project 1 Open the Thermal Profile screen 2 In the Experiment
2. If you need the new plateau to be the first plateau in the segment skip step 2 and go directly to step 3 Right click within the same segment A short cut menu opens Click Add Plateau The program adds a new plateau immediately after the selected plateau or to the start of the segment if no plateau is selected The new plateau has a 25 C temperature and a 30 second duration Edit the temperature and duration of the new plateau as needed See the instructions above To edit a plateau temperature and To edit a plateau duration To remove a plateau 1 On the display select the plateau that you want to remove To select a plateau click directly on it The plateau becomes red in the display Right click on the segment containing the selected plateau A short cut menu opens Click Remove Plateau The program deletes the selected plateau from the thermal profile Agilent AriaMx Real Time PCR Program Setting Up the Thermal Profile 7 Edit segments To add a segment 1 On the display hover your cursor over an existing segment that will be adjacent to the new segment 2 Click the icon shown below on either the left side or right side of the existing segment T Clicking the icon on the left adds the new segment just before the existing segment Clicking the icon on the right adds the new segment just after the existing segment The program opens a placeholder for the new segment shown below listing th
3. m oe c 0 0 ee o 0 an AriaMx Real Time PCR Software User Manual For Research Use Only Not for Use in Diagnostic Procedures S Agilent Technologies Notices Agilent Technologies Inc 2014 No part of this manual may be reproduced in any form or by any means including elec tronic storage and retrieval or translation into a foreign language without prior agree ment and written consent from Agilent Technologies Inc as governed by United States and international copyright laws Manual Part Number 68830 90000 Edition Revision AO July 2014 Agilent Technologies Inc 5301 Stevens Creek Blvd Santa Clara CA 95051 Warranty The material contained in this docu ment is provided as is and is sub ject to being changed without notice in future editions Further to the max imum extent permitted by applicable law Agilent disclaims all warranties either express or implied with regard to this manual and any information contained herein including but not limited to the implied warranties of merchantability and fitness for a par ticular purpose Agilent shall not be liable for errors or for incidental or consequential damages in connec tion with the furnishing use or per formance of this document or of any information contained herein Should Agilent and the user have a separate written agreement with warranty terms covering the material in this document that conflict w
4. 1 From the toolbar click File gt Logout The program logs you out and the Login dialog box opens In the Login dialog box click Change Password The Change Password dialog box opens Type your Username Old Password and New Password into the fields Type the new password again into the Confirm Password field Passwords must be 6 15 alphanumeric characters in length and include at least one number Click OK The Change Password dialog box closes and you are returned to the Login dialog box Type your Username and Password into the fields using your new password Click Login Agilent AriaMx Real Time PCR Program 293 16 Help for the AriaMx ET Electronic Tracking Software Create a multiple experiment analysis project in the AriaMx ET software Multiple experiment analysis MEA is a feature in the AriaMx software that allows you to display and analyze the results from two or more post run experiments together in a single file called a project See Overview of multiple experiment analysis on page 235 for a description of MEA Although the ET version of the AriaMx software allows you to create MEA projects and analyze the data in those projects the projects are not stored in a database and the program does not electronically track the actions taken on projects To create an MEA project in the AriaMx ET program 1 Export the post run experiments to be added to the project from the database to a file system S
5. C Rox C Hex O Cie 0 os O ser occ 0 06 Agilent AriaMx Real Time PCR Program Setting Up the Plate 6 To assign dyes and target names 1 On the Plate Setup screen select all the wells in the plate map that contain the same target For instructions on well selection see Select and view wells in the plate map on page 74 2 Inthe Properties panel under Add Dyes mark the Use check box for the dye used for target detection in the selected wells 3 If the fields for entering target names are not displayed click the arrow next to Targets The fields appear to the right of the dye names 4 For the marked dye type a name into the adjacent Target Name field The program assigns the target name to the selected wells If you do not assign a target name the program uses the dye name as the target name 5 Repeat steps 1 4 for all wells included in use in the experiment 6 Optional Select a new color to associate with a target a Click the colored dot to right of the Target Name field b In the selection box that opens click the desired color or click Advanced for more color options Select a reference dye You can include a reference dye e g ROX dye to normalize the fluorescence signal of the reporter dye To assign a reference dye e On the Plate Setup screen select the reference dye from the Reference Dye drop down list in the Properties panel The program assigns the target name RE
6. Set analysis criteria for a project on page 248 278 Agilent AriaMx Real Time PCR Program How To Examples for Multiple Experiment Analysis Projects 15 Step 4 View the initial template quantity calculations in the standard curve To view the standard curve open the Graphical Displays screen If the Standard Curve graph is not already displayed click the Standard Curve icon at the bottom of the screen In the right panel next to Fluorescence Term select the fluorescence data type to be used for analysis Then next to Compare select Target When compared by target the program analyzes the data across experiments by target name The Cq values from the Unknown wells for your target of interest are plotted on the same graph as the standard curve for this target that was generated from the Standard wells run on the other experiment You can move your cursor over the data point for an Unknown well or replicate set to view the initial template quantity calculated for the target in that well or replicate set see below Standard Curve Target cq GR 2 Comparative Quantitation _Unknowns BS Replicate 18 Target A Initial Template Oty 1 52 7088E 000 Quantification Cycle Cg 24 40 5 By 2 Initial Quantity relative Agilent AriaMx Real Time PCR Program 279 15 Example 2 280 How To Examples for Multiple Experiment Analysis Projects How to normalize target quantities in Unknown
7. on page 223 you can generate the report file By default reports are saved to the folder C Users Public Documents Agilent AriaMx Reports but you can select a different folder when you generate the report Agilent AriaMx Real Time PCR Program Generating Reports and Exporting Results 11 To generate the report 1 At the bottom of the Generate Report screen click Generate Report The Save As dialog box opens 2 Specify a file name and folder for the report file and click Save The program generates the report and then opens it in the appropriate application either Microsoft PowerPoint or your default PDF reader Configure the report The program provides numerous ways for you to customize the report configuration It also allows you to load a report configuration definition to quickly configure the report to a set of previously saved settings Load a saved report configuration definition If you already have a saved report configuration definition on your system that you want to use for the current experiment you can load that definition from the Generate Report screen The saved definition must be for the same experiment type The program comes preloaded with a default report configuration definition that is automatically loaded You can also configure your own report and save the configuration for later use see Create or edit report configuration definitions on page 226 To load a report configuration definitio
8. Compare standard curves in a project on page 260 Compare Relative Quantity charts in a project on page 262 Compare Allele Determination graphs in a project on page 264 When all experiments in the project are displayed the Graphical Displays screen shows one type of graph at a time However if you set the toggle button to display only one experiment the screen can show multiple graphs for that one experiment similar to when a single experiment file is open By default data from all of the wells that you selected on the Analysis Criteria screen are included in the analysis and displayed in the graphs You can limit the graphs to only display data from particular wells or replicate sets using the check boxes in the result table The result table is described below Agilent AriaMx Real Time PCR Program 251 13 Analyzing Multiple Experiment Analysis Project Results 252 Result table The result table on the right side of the Graphical Displays screen shows results for each well if replicates are treated individually or replicate set if replicates are treated collectively that you selected on the Analysis Criteria screen see Select the wells and well types to include in analysis on page 248 Row selection You can select individual rows within the results table to limit the graphs to displaying data only from particular wells replicate sets Click directly on a row to select it Press Ctrl to select multiple
9. Overview of the user interface Home screen The Home screen provides an introduction to the AriaMx system and a list of program features See Introduction to the AriaMx software on page 17 for detailed information on the Home screen Menu toolbar At the top of the program window are the File and Instrument menus e The File menu contains commands for opening closing saving and printing experiments and projects e The Instrument menu contains commands for connecting to configuring exporting data from and testing the AriaMx instrument Tabs The user interface of the AriaMx program allows you to open up to 5 experiments at a time or one project at a time The program displays each open experiment or project on its own tab enabling you to quickly switch between them Click the icon to the right of the tabs to open a new tab New tabs open to the Getting Started screen Left and right panels When you are working in an experiment or project the left side of the screen displays the Experiment Area panel and the right side of the screen displays a panel with tools for the currently open screen e g the Report Configuration panel is displayed on the Generate Report screen You can hide these panels by clicking the arrow icon next to the panel name Click the arrow icon again to expand the panel Agilent AriaMx Real Time PCR Program 19 1 20 Getting Started with the AriaMx Program Home Notifications Help i
10. The program assigns the HCP to the experiment and calibrates the melt data accordingly To assign a recently used HCP 1 On the Analysis Criteria or Graphical Displays screen click the arrowhead next to the HCP icon at the bottom of the screen HEP In the menu that opens hover your cursor over Recently Used Data A list of recently used HCPs opens Click the HCP that you want to assign to the current experiment The program assigns the HCP to the experiment and calibrates the melt data accordingly Agilent AriaMx Real Time PCR Program Setting Analysis Criteria 9 To clear an HCP assignment 1 On the Analysis Criteria or Graphical Displays screen click the arrowhead next to the HCP icon at the bottom of the screen HCP 2 Inthe menu that opens click Clear Calibration Data The experiment no longer has an HCP assigned If desired you can now assign a different HCP to the experiment Agilent AriaMx Real Time PCR Program 165 9 Setting Analysis Criteria Assign an HRM calibration plate 166 Agilent AriaMx Real Time PCR Program 10 Viewing Graphical Displays of the Results Overview of the Graphical Displays screen 168 Graphs 168 Result table 169 Display options 169 Zooming 1 1 View the Amplification Plots 173 View the Melt Curve Raw Derivative Curve 183 View the Melt Curve Difference Plots 189 View the Standard Curve 196 View the Relative Quantity 201 View the Allele Determination graph 206 Cu
11. Creating Opening an Experiment 4 Save a copy of an existing experiment You can use the Save As command to copy the open experiment and save it with a new experiment name To copy an existing experiment using the Save As command 1 Open the existing experiment that you want to copy 2 Click File gt Save As The Save As dialog box opens 3 Select a folder for the new experiment and type a name into the file name field 4 Click Save The program saves the open experiment under the new name Agilent AriaMx Real Time PCR Program 49 4 Creating Opening an Experiment Create a template from an existing experiment You can use the Save As Template command to create a template file based on the plate setup and thermal profile of an existing experiment You can later use the template to quickly create new experiments See Create an experiment from a template on page 47 To create a template from an existing experiment using the Save As Template command 1 Open the existing experiment that you want to create a template from 2 Click File gt Save As Template The Save As dialog box opens with the file type set to AriaMx Template Files amxt 3 Select a folder for the new template and type a name into the file name field 4 Click Save The dialog box closes and the program saves the new template file amxt to the designated folder 50 Agilent AriaMx Real Time PCR Program Creating Opening an Experiment 4
12. Setting Up the Plate Assign dyes targets Use the check boxes drop down lists and fields under Assign Dyes Targets to indicate which dyes are being used in each well and what target each dye is detecting Dye assignments are required but target name assignments are optional If different wells will be using the same dye to detect different targets assigning a unique name to each target enables the program to treat each target separately during analysis Add Dyes Targets 4 Use Dye Name Target Name C FAM C Rox DO O Ga C cvs C sen To assign dyes and target names oc coco 06 1 On the Plate Setup screen select all the wells in the plate map that contain the same target For instructions on well selection see Select and view wells in the plate map on page 74 2 Under Add Dyes mark the Use check boxes for the dyes used for target detection in the selected wells 3 Ifthe fields for entering target names are not displayed click the arrow next to Targets The fields appear to the right of the dye names 4 For the marked dyes type a name into the adjacent Target Name field The program assigns the target names to the selected wells If you do not assign a target name to a marked dye the program uses the dye name as the target name 5 Repeat steps 1 4 for all wells included in use in the experiment 6 Optional Select a new color to associate with a target a Click the colored dot to ri
13. Software 16 View transaction logs in the AriaMx ET software If you are running the electronic tracking ET version of the AriaMx software and you logged in using an administrator account you can view the transaction logs which track archiving and restoring activities To open the Transaction Log dialog box At the top of the program window click Admin gt Transaction Log Fij Transaction Log Database i Archive Expenment Name Archived To Database Archived On Comparative Quantitation Multiplex Example Analarchive Uz Apr 2014 17 59 39 HCP file for HRM SNP IV Example Ania larchive 02 Apr 2014 11 59 40 View transaction logs for primary database The Transaction Log dialog box can display all the experiments that have been archived from the primary database Note that the logs do not include archived experiments that were later restored to their original primary database To view the log of archive transactions 1 Open the Transaction Logs dialog box Admin gt Transaction Log 2 Next to Database select Primary if not already selected Agilent AriaMx Real Time PCR Program 315 16 Help for the AriaMx ET Electronic Tracking Software The table lists the experiments that have been archived Experiment Name column which database they were archived to Archived To Database column and the date and time that they were archived Archived On column To help locate a particular experiment type a search term into th
14. Temperature C To use HRM analysis for SNP genotyping your experiment needs to include positive control samples for each base pair possibility homozygous as well as heterozygous positive control samples Then when viewing the difference plots designate the target in one of the homozygous positive control samples as the control target You can then compare samples of unknown genotype to the samples of known genotype to visually determine the correct genotype group for the target in the unknown sample Agilent AriaMx Real Time PCR Program Viewing Graphical Displays of the Results 10 Select a fluorescence data type For the Y axis of the difference plots you can select to use raw or normalized fluorescence values To select the type of fluorescence data displayed in the Difference Plots 1 Select the Melt Curve Difference Plots graph on the Graphical Displays screen 2 Inthe right panel select one of the options next to Fluorescence Term The possible options are R Raw fluorescence Rn Normalized fluorescence normalized to reference dye Assign the control target The values plotted on the Y axis are the difference in fluorescence values between a target in a particular well or replicate set and a specified control target You can designate the control sample and target using the using the tools in the panel on the right side of the screen Typically a target from a homozygous positive control sample is selected
15. Type of cycles Amp 2 Step Amplification using 2 plateaus one for denaturation and 50 one for annealing extension Amp 3 Step Amplification using 3 plateaus one for denaturation one 50 for annealing and one for extension Melt dissociation of PCR products tT Hot Start Hot start activation of PCR enzyme UDG DNA Uracil DNA glycosylation reaction in a reaction in which UNG uracil N glycosylase was used to digest amplicon carryover from a previous PCR amplification you can add this segment to the beginning of the thermal profile to heat inactivate the UNG enzyme Reverse transcription of RNA into DNA 130 Agilent AriaMx Real Time PCR Program Setting Up the Thermal Profile 7 Data Collection Marker A data collection marker is a magnifying glass icon that indicates the points designated for fluorescence data collection by the instrument For data collection markers on a plateau the instrument collects fluorescence data at the end of the plateau duration The minimum duration for a plateau that includes data collection is 3 seconds For data collection markers on a melt ramp only melt segment ramps can accept collection markers the instrument collects fluorescence data throughout the ramp period Edit the thermal profile From the Thermal Profile screen you can edit any element of the thermal profile including adding and deleting segments and plateaus and changing plateau temperatures and durations Ed
16. in any given well and the average Tm for the plate When you associate your Allele Discrimination experiment with an HCP experiment the program Agilent AriaMx Real Time PCR Program Selecting an Experiment Type 5 subtracts the offset temperature from the calculated Tm in each well This normalization improves the accuracy and clarity of the difference plots and the raw derivative melt curves In order to calculate the offset temperatures an HCP must contain the identical amplicon in each well During the HCP run that amplicon is melted in an HRM segment The program then calculates a Tm for each well and an average Tm for the plate All HCP experiments must be set up and run directly from the instrument you cannot set up an HCP experiment from your PC See Run an HRM calibration plate HCP on page 35 for instructions You can use the same HCP experiment for multiple Allele Discrimination experiments For each AriaMx instrument Agilent recommends running a new HCP experiment at least once per year Well types for Allele Discrimination DNA Binding Dye experiments Unknown Contains a complete reaction mixture including a test template that contains an unknown amount of the specific target No template control contains all reaction components except the template nucleic acid This control is useful for detecting amplicon contamination Homo Allele A Contains a complete reaction mixture with a positive control templat
17. next to Report Type select PDF to select a PDF report or select PowerPoint to select a PowerPoint report Generate the report After you configure the report as desired see the tasks under Configure the report you can generate the report file By default reports are saved to the folder C Users Public Documents Agilent AriaMx Reports but you can select a different folder when you generate the report To generate the report 1 At the bottom of the Generate Report screen click Generate Report The Save As dialog box opens 266 Agilent AriaMx Real Time PCR Program Generating Multiple Experiment Analysis Reports and Exporting Results 14 2 Specify a file name and folder for the report file and click Save The program generates the report and then opens it in the appropriate application either Microsoft PowerPoint or your default PDF reader Configure the report The program provides numerous ways for you to customize the report configuration It also allows you to load a report configuration definition to quickly configure the report to a set of previously saved settings Load a saved report configuration definition If you already have a saved report configuration definition on your system that you want to use for the current project you can load that definition from the Generate Report screen The saved definition must be for the same experiment type The program comes preloaded with a default report configurat
18. the program uses the following guidelines for normalizing the data for a target of interest e If anormalizer target is designated in the same well as the target of interest the program uses that normalizer for normalization e If anormalizer target is not found in the same well as the target of interest the program uses a normalizer from wells of the same sample name within the same experiment e If anormalizer target is not found in wells of the same sample name within the same experiment the program uses a normalizer from wells with the same sample name that were run on a different experiment in the project To compare by target 1 Select the Relative Quantity chart on the Graphical Displays screen 2 Inthe right panel next to Compare select Target Consolidate the relative quantities When you select to consolidate the relative quantities the program displays all of the quantities from all experiments on a single chart The program calculates the relative quantities in the same manner as it does when you compare the data by target To consolidate the relative quantities 1 Select the Relative Quantity chart on the Graphical Displays screen 2 Inthe right panel next to Compare select Consolidated Agilent AriaMx Real Time PCR Program 263 13 Analyzing Multiple Experiment Analysis Project Results Compare Allele Determination graphs in a project For projects comprised of Allele Discrimination experiments or User Def
19. 179 10 Viewing Graphical Displays of the Results 180 Adjust threshold fluorescence values by altering the algorithm settings The default threshold fluorescence values calculated by the program are background based thresholds Background based thresholds are based on the level of fluorescence noise in the background The program calculates the background noise from the levels of fluorescence present in the selected wells the wells included in the analysis during the early cycles of PCR before product begins to accumulate and the fluorescence levels rise By default the cycles that the application uses for calculating the background noise are cycles 5 through 9 To calculate the threshold fluorescence values the program then multiplies the standard deviation sigma of the raw fluorescence in the background cycles by a mathematical constant known as a sigma multiplier The default sigma multiplier is 10 You can adjust the cycles used for calculating background noise and the sigma multiplier used in the algorithm from the Graphical Displays screen Adjusting these settings results in a change to the threshold fluorescence values To change the range of cycles used for determining background noise 1 Select the Amplification Plots graph on the Graphical Displays screen 2 Inthe right panel click the downward arrowhead above the result table to display the advanced analysis parameter settings The current cycle range is displayed in
20. As dialog box select a different folder and or file name 2 Click Start to start the test Agilent AriaMx Real Time PCR Program 3 33 3 34 Performing Hardware and Software Tests and HRM Calibrations The program displays the status of the test in the Test Log area of the dialog box When finished the program updates the Status column in the dialog box and displays the overall result Passed or Failed If the test failed contact Agilent Technical Support for assistance See Contact Agilent Technical Support on page 327 3 Click Open Report to open the PDF report generated by the program 4 To run the test again click Reset to clear the results of the previous test The Operational option on the Qualifications dialog box has tools to run an Operational Qualification OQ test However the Operational option is only available to Agilent Service Engineers with an appropriate password Agilent AriaMx Real Time PCR Program Performing Hardware and Software Tests and HRM Calibrations 3 Run an HRM calibration plate HCP For experiments that include a high resolution melt HRM segment before you can analyze the HRM data on a Difference Plots graph you must associate the experiment with an HRM calibration plate HCP that was run on the same instrument The purpose of the HCP is to calculate an offset temperature for each well in order to help normalize well to well temperature variations See About HRM calib
21. Configuration Panel of the Generate Report screen under Header amp Footer mark the check boxes for the pieces of information that you want to include in the header or footer Clear the check boxes for any pieces of information that you do not want to include If marked the Project Name and Analysis Date appear in the header of the report The Page Number if marked appears in the footer of the report Agilent AriaMx Real Time PCR Program 269 14 Generating Multiple Experiment Analysis Reports and Exporting Results 270 Rearrange the pages of the report You can set the order of the items included in the report by dragging and dropping within the report preview To rearrange the pages 1 At the bottom of the Generate Report screen click Rearrange The program adjusts the display of the report preview to show thumbnails of all items included in the report with a number next to each item to indicate its order in the report Note that some items take up more than one page For those items the number of pages is indicated in parentheses after the item name 2 For an item that you want moved to a different order click and drag on the thumbnail Drop the item into the desired order Repeat for any other items you want to rearrange 3 Click Rearrange again The program sets the display of the report preview back to the standard mode with the pages displayed in the new arrangement Create or edit report configuration definiti
22. Convert an experiment to a new experiment type The Convert Experiment Type command can convert a post run experiment into another experiment type This command is useful when the experiment was set up as one type and the data needs to be re analyzed as a different experiment type The program applies the analysis algorithms and display options based on the new experiment type To convert an experiment to a new experiment type 1 Open the existing post run experiment that you want to convert 2 Click File gt Convert Experiment Type and in the sub menu select the new experiment type A message box opens notifying you that the conversion was successful and that some well types may have changed 3 Click OK to close the message box The program creates a new experiment file for the converted experiment and opens the experiment to the Plate Setup screen By default the new experiment has the same name as the parent experiment with the word Converted added at the beginning 4 Click File gt Save As The Save As dialog box opens 5 Select a folder for the new experiment Type a name into the file name field or use the default file name 6 Click Save The program saves the new experiment to the designated folder Agilent AriaMx Real Time PCR Program 51 4 Creating Opening an Experiment Convert an experiment to a new experiment type 52 Agilent AriaMx Real Time PCR Program Selecting an Experiment Type Overview of Exp
23. Curve graph on the Graphical Displays screen 2 Inthe right panel click the downward arrowhead above the result table to display the advanced analysis parameter settings 3 In the Max Number field below the Product Melting Temperature heading type in the desired maximum number of products or click the buttons to adjust the number of products in the field to the desired value The default value is 4 The highest value allowed is 6 Product Melting Temperature Max Number 4 To set a minimum peak height for an amplification product 1 Select the Melt Curve Raw Derivative Curve graph on the Graphical Displays screen 2 Inthe right panel click the downward arrowhead above the result table to display the advanced analysis parameter settings 3 Next to Min Peak Height select On to turn on the minimum peak height option A new field appears for entering the minimum peak height and a horizontal line appears on the graph marking the minimum height Min Peak Height On Off 0 170 ry Agilent AriaMx Real Time PCR Program 187 10 Viewing Graphical Displays of the Results 4 In the field type the desired minimum peak height or click the buttons to adjust the value in the field Clicking the Reset icon sets the Minimum Peak Height value to the lowest peak among the set of highest peaks across all selected wells The height of the horizontal line on the graph adjusts accordingly and the program only counts a peak
24. Horizontal if the replicate reactions will be arranged horizontally in rows e Select Vertical if the replicate reactions will be arranged vertically in columns In the Wells per replicate set field type the number of replicate wells per reaction or click the buttons to enter the desired number Agilent AriaMx Real Time PCR Program Setting Up the Plate 6 The assigned replicate number appears in each selected well 5 If desired make adjustment to the auto assignments in any of the wells of the plate by switching to the Manual option and manually assigning replicate numbers to those wells To manually assign replicates 1 On the Plate Setup screen select a set of wells that are part of the same replicate set Make sure that the selected wells are of the same well type and contain identical targets For instructions on well selection see Select and view wells in the plate map on page 74 2 Inthe Properties panel under Replicates select Manual if not already selected Replicates ECTE Auto Assign Replicate l Humber Auto Increment 3 Inthe Assign Replicate Number field type in the desired replicate number for the selected wells or click the buttons to enter the desired number The assigned replicate number appears in each selected well To assign replicates using Auto Increment 1 On the Plate Setup screen assign well types as
25. In the center of the Plate Setup screen is a representation of the wells of a 96 well plate This diagram called a plate map is used for showing which wells in the plate are in use in the current experiment what kind of sample is in each well which dyes are being used for detection in the wells and what targets those dyes are detecting The properties assigned to the wells are indicated in the plate map To see more detailed information on the properties of a particular well such as target names and standard quantities hover your cursor over the well Standar Standard Well type or Well name Standard quantty Replicate number Reference dye You can also open a mini plate map to view the details in a selected set of wells Well type All wells that are in use in the experiment need to be assigned a well type This assignment indicates to the program the type of reaction in the well For example the Unknown well type is used for experimental templates in which the quantity of the target s is unknown When the Show setting on the Plate Setup Properties panel is set to Type the well type appears at the top of the well The available well types vary depending on the experiment type Well name Sample name If desired you can assign names to the wells of the plate When the Show setting on the Plate Setup Properties panel is set to Name the well name appears at the top of the well The Comparative Quantitation Allel
26. Password eeeesesscesscssceesseescoeeeeeeeee Retype Password User Type Change Password encima aba Create a user account Create user accounts for each user who needs to log in to the AriaMx ET program and access the database To create a user account 1 Open the User Management dialog box to the User Settings tab 2 Click Create User The fields in the bottom panel of the dialog box e g Username Full Name etc become editable 3 Complete all fields Username Type a username for the new account Full Name Type the name first and last of the person who will be using the account Agilent AriaMx Real Time PCR Program 299 16 300 Help for the AriaMx ET Electronic Tracking Software Password Type a password for the account must be 6 15 alphanumeric characters in length and include at least one number Retype Password Type the same password you entered above 4 Next to User Type select the access privileges for the new user e Select Administrator to give the user administrator privileges e Select User to give the user standard privileges Administrator privileges include managing user accounts and databases archiving and restoring experiments and viewing audit trails and transaction logs 5 Click Close to close the dialog box and apply your changes Disable or enable a user account Once created you cannot delete a user account but you can disable the account to prevent the user from bein
27. Plate nanograms To assign the standard quantities for a target in the Standard wells 1 On the Plate Setup screen select the Standard wells For instructions on well selection see Select and view wells in the plate map on page 74 2 Inthe Properties panel under Standard Quantities select which target in the selected wells is the standard target i e the target of known quantity in the template 86 Agilent AriaMx Real Time PCR Program Setting Up the Plate 3 Inthe Starting Amount field type in the quantity of the standard target present in the first replicate set of Standard wells You will be asked to specify the units for this amount step 5 This quantity must be either the highest quantity or the lowest quantity in the dilution series of the standard template sample 4 Inthe drop down list labeled A factor of select the dilution factor used to generate the dilution series of the standard template For example if each standard quantity is separated by a factor of 10 select 10x Negative dilution factors are used to specify a decrease in quantity from the starting amount while positive dilution factors specify an increase from the starting amount 5 In the drop down list labeled Units for Plate select the units of the quantity entered in the Starting Amount field Note that all Standard wells on the plate must use the same units To clear the assigned standard quantity from one or more wells e Select the
28. Real Time PCR Program Viewing Graphical Displays of the Results 10 Adjust the graph properties You can adjust certain properties of the Amplification Plots graph e g the scales of the axes the graph title and the background color through the short cut menu and the Graph Properties dialog box See Customize graph properties on page 211 for more information Adjust the baseline correction settings By default the program automatically determines which cycles to use to calculate the baseline in order to produce baseline corrected fluorescence AR data for each well target combination For each target in each well the raw fluorescence data over a specific range of cycles are fit to a line using a linear least mean squares algorithm to produce a baseline The value of the baseline function is calculated for every cycle and subtracted from the raw fluorescence to produce baseline corrected fluorescence AR You can view and adjust these software determined cycle ranges known as adaptive values from the Baseline Correction dialog box You can adjust the cycle range for individual plots or set all plots to the same cycle range To open the Baseline Correction dialog box 1 Select the Amplification Plots graph on the Graphical Displays screen 2 In the right panel click the downward arrowhead above the result table to display the advanced analysis parameter settings 3 Next to Baseline Correction click Adjust An asterisk
29. Relative Quantity chart is not already displayed click the Relative Quantity icon at the bottom of the screen In the right panel next to Fluorescence Term select the fluorescence data type to be used for analysis Then next to Compare select Target When compared by target the program analyzes the data across experiments by target name and generates a separate Relative Quantity chart for each target The program normalizes the data for the target of interest using the normalizer from the same sample You can hover your cursor over a bar on the chart for the target of interest to view the relative quantity calculated for the target in that well or replicate set The program generates a graph for the normalizer target but no data are plotted on it Agilent AriaMx Real Time PCR Program 281 15 How To Examples for Multiple Experiment Analysis Projects Example 2 Relative Quantity Target E 2 Comparative Quantitation_Unknowns B9 Replicate 19 Target B ARn B9 6 9446 a 2 i E Z F F E F f a S F F s amp F amp Well ID Replicate Relative Quantity Norm No relative quantity data available x Relative Quantity AR Well ID Replicate 282 Agilent AriaMx Real Time PCR Program 16 Help for the AriaMx ET Electronic Tracking Software Overview of the AriaMx ET software 284 Open an experiment in
30. Setup screen Agilent AriaMx Real Time PCR Program 287 16 Help for the AriaMx ET Electronic Tracking Software To open an earlier snapshot of an experiment from the File menu 1 From the toolbar click File gt Open The Open Experiment dialog box opens with a list of all experiments in the primary database organized by user see image above 2 Expand the node for the desired experiment to view the snapshots available for the experiment The date and time stamp for the snapshot are listed in the Date column 3 Click a snapshot to select it then click Open The dialog box closes and the program opens the experiment to the Plate Setup screen 288 Agilent AriaMx Real Time PCR Program Help for the AriaMx ET Electronic Tracking Software 16 Import and export experiments in the AriaMx ET software AriaMx ET program tracks the actions taken on a post run experiment using a primary database with controlled access If you want to open a post run experiment that is saved to a local or network folder you first must import the experiment into the database Similarly if you want to make an experiment available outside of the database you must first export it to a local or network folder Import experiments into the database Importing a post run experiment into the database creates a controlled copy of the experiment and makes it available for opening in the AriaMx ET program To import an experiment from a folder into the data
31. Under Threshold Fluorescence locate the target that you want to adjust If the lock icon next to the target is in the locked position click on it to unlock it On the graph hover your cursor over the triangle marker for the threshold line that you want to adjust The cursor becomes a vertical double side arrow and a tooltip box opens displaying the name of the target and its current threshold fluorescence value Agilent AriaMx Real Time PCR Program Viewing Graphical Displays of the Results 10 Amplification Plots S S 8 gg Fluorescence AR o CY5 65 000 5 Click and drag the triangle marker to the desired position on the Y axis The program sets the threshold fluorescence value for that target its new Y axis value The program also updates the value listed for that target in the right panel To reset the threshold fluorescence value for a target back to the default value calculated by the program 1 Select the Amplification Plots graph on the Graphical Displays screen 2 Inthe right panel click the downward arrowhead above the result table to display the advanced analysis parameter settings 3 Under Threshold Fluorescence click the Recalculate icon a next to the desired target The program resets the threshold fluorescence value in the field back to the default value and adjusts the position of the corresponding horizontal line on the Amplification Plots graph Agilent AriaMx Real Time PCR Program
32. View the Melt Curve Difference Plots 189 About difference plots 189 Select a fluorescence datatype 191 Assign the control target 191 View data fora single data point 192 Manually assign Unknowns to a genotype call 192 Adjust the graph properties 195 View the Standard Curve 196 View data for a single data point 196 Select a fluorescence datatype 197 View the R squared values slopes and amplification efficiencies 198 Adjust the graph properties 199 Manually adjust threshold fluorescence values 199 Display and adjust confidence intervals 200 Agilent AriaMx Real Time PCR Program View the Relative Quantity 201 About relative quantities 201 Select a fluorescence datatype 202 Set the Y axis scale for the Relative Quantity chart 202 Add error bars to the Relative Quantity chart 203 Adjust the graph properties 203 Select the algorithm method 203 Enter the amplification efficiencies for the targets 204 View the Allele Determination graph 206 View data for a single data point 206 Select data type and fluorescence type to display 207 Display genotype groups onthe graph 208 Adjust the graph properties 209 Adjust the last cycle 209 Rename the genotype groups 210 Customize graph properties 211 Customize graph properties using the short cut menu 211 Customize graph properties using the Graph Properties dialog box 11 Generating Reports and Exporting Results 221 Generate report of results 222 View a preview of the report 222 Select report
33. a new experiment from a template the experiment has the plate setup and thermal profile of the selected template After you create the experiment you can edit the plate setup and thermal profile as desired The AriaMx software comes with three sample template files The templates are available in the folder C Users Public Documents Agilent AriaMx Experiment Templates To create an experiment from a template 1 Open the Getting Started screen 2 Under New Experiment click My Templates The center of the screen displays the template files in the default template folder You can toggle between displaying the templates in list format and tile format by clicking the icons in top right corner List view icon aie Tile view icon 3 In the Experiment Name field type a name for the new experiment 4 Select the desired template and create the experiment e If the template is in the default folder click directly on the template to select it and then click Create or double click directly on the template The program creates the new experiment and opens the experiment to the Plate Setup screen e If the template is not in the currently selected folder click the Browse to Template icon shown below to open the browser window Browse to the folder of the desired template file Select the file and click Open The program creates the new experiment and opens the experiment to the Plate Setup screen Bo Agilent AriaMx Real Ti
34. all samples to be included in the experiment Assign dyes targets Use the check boxes drop down lists and fields under Assign Dyes Targets to indicate which dyes are being used in each well and what target each dye is detecting Dye assignments are required but target name assignments are optional If different wells will be using the same dye to detect different targets assigning a unique name to each target enables the program to treat each target separately during analysis Add Dyes Targets 4 Use Dye Name Target Name O a o 0 Rox x 0e 0 Gin 0e O eer 0c 0 0 106 Agilent AriaMx Real Time PCR Program Setting Up the Plate 6 To assign dyes and target names 1 On the Plate Setup screen select all the wells in the plate map that contain the same target For instructions on well selection see Select and view wells in the plate map on page 74 2 Under Add Dyes mark the Use check box for the dye used for target detection in the selected wells 3 Ifthe fields for entering target names are not displayed click the arrow next to Targets The fields appear to the right of the dye names 4 For the marked dye type a name into the adjacent Target Name field The program assigns the target name to the selected wells If you do not assign a target name the program uses the dye name as the target name 5 Repeat steps 1 4 for all wells included in use in the experiment 6 Optional Select a new color to
35. an instrument since last launching the application the Login dialog box opens Select your Username from the drop down list type your login password into the Password field and click Login After logging in the File Explorer dialog box opens 4 In the list of folders on the left side of the File Explorer dialog box browse to the folder of the experiment on the instrument The root folder for the experiment is the folder for the user who ran the experiment The subfolder is named for the experiment type 5 Click directly on the experiment for which you want to transfer the run data 6 Click Copy amp Delete Agilent AriaMx Real Time PCR Program Running and Monitoring Experiments 8 The program transfers the post run experiment file from the instrument to the default experiment storage folder on your PC the program deletes the file from the instrument in the process If the pre run experiment is already saved to your PC the program saves the post run experiment file with a bracketed number appended to the end of the file name e g Experiment1 1 7 Open the experiment file in the AriaMx program to view the results of the run Retrieve data using a USB drive You can connect a USB drive to the instrument and save the data to the drive and then transfer it to your PC This approach is the only way to retrieve data from an instrument that is not network connected 1 Insert an external USB drive into the USB port on th
36. and Calibrator wells using a normalizer target from a separate experiment The Comparative Quantitation experiment type is ideal for comparing amounts of mRNA of a target of interest in treated vs untreated or normal vs diseased cells or tissues In these studies the control sample is referred to as the calibrator and the test samples are referred to as the unknown samples To help correct for spurious differences in the level of a target of interest that are not due to the experimental condition being tested it is important to amplify a normalizer target from each calibrator and unknown sample An ideal normalizer target is one that you know is not differentially expressed as a result of your experimental conditions e g a housekeeping gene In a MEA project composed of Comparative Quantitation experiments or User Defined experiments designed to determine relative quantities the data for a target of interest can be normalized to data from a normalizer target that was run in a separate experiment This example walks you through the process for setting up a project in which the target of interest and normalizer targets are on different experiments Step 1 Create the Project Create the project and make sure to add the two experiments to the project the experiment in which the target of interest was amplified in the unknown and calibrator samples and the experiment in which the normalizer target was amplified in the same unknown and cali
37. as the target name Repeat steps 1 4 for all wells included in use in the experiment Optional Select a new color to associate with a target a Click the colored dot to right of the Target Name field b In the selection box that opens click the desired color or click Advanced for more color options Select a reference dye You can include a reference dye e g ROX dye to normalize the fluorescence signal of the reporter dye To assign a reference dye e On the Plate Setup screen select the reference dye from the Reference Dye drop down list in the Properties panel The program assigns the target name REF to all wells in use in the experiment and displays an R in the wells of the plate map to indicate that the well contains a reference dye Agilent AriaMx Real Time PCR Program Setting Up the Plate 6 Assign Alleles Use the drop down lists under Allele to indicate which target is to be designated as Allele A and which target is Allele B To assign alleles 1 In the Dye Target Name drop down list for Allele A select the target that represents allele A 2 Inthe Dye Target Name drop down list for Allele B select the target that represents allele B Assign replicates Replicates are wells that contain identical reaction components repeats You can assign replicates using the Manual option or the Auto option When you designate replicate wells on the Plate Setup screen you can set the analysis criteria
38. as the control To assign the control target 1 Select the Melt Curve Difference Plots graph on the Graphical Displays screen 2 Inthe right panel click the downward arrowhead above the result table to display the Control Target settings 3 Under Control Target use the drop down lists to specify the well or replicate set that contains the control sample and the target in that well replicate set that is detecting the control target a Inthe Well Type drop down list select the well type in which the control target was amplified Typically one of the homozygous control wells is selected Homo Allele A or Homo Allele B b In the Replicate or Well Id drop down list select the specific well or replicate set in which the control target was amplified c In the Target drop down list select the dye or target name for the control target Agilent AriaMx Real Time PCR Program 191 10 Viewing Graphical Displays of the Results 192 View data for a single data point Each curve on the graph is the plot for a single target in a single well or replicate set Each data point that makes up the curve is a fluorescence value for that target well measured at a particular temperature plotted on the X axis The program connects these data points to draw the raw derivative curves displayed on the graph To view a summary of the data for a single data point on a plot 1 Select the Melt Curve Difference Plots graph on the Graphical Displ
39. associate with a target a Click the colored dot to right of the Target Name field b In the selection box that opens click the desired color or click Advanced for more color options Select a reference dye You can include a reference dye e g ROX dye to normalize the fluorescence signal of the reporter dye To assign a reference dye e On the Plate Setup screen select the reference dye from the Reference Dye drop down list in the Properties panel The program assigns the target name REF to all wells in use in the experiment and displays an R in the wells of the plate map to indicate that the well contains a reference dye Agilent AriaMx Real Time PCR Program 107 6 Setting Up the Plate Assign replicates Replicates are wells that contain identical reaction components repeats You can assign replicates using the Manual option or the Auto option When you designate replicate wells on the Plate Setup screen you can set the analysis criteria to average results from those wells or treat the wells separately When assigning replicates if you see a flashing red warning Icon in the Properties panel next to Replicates you have an invalid replicate set on the plate To be valid all the wells of a set must be of the same well type and have the same target assignments Hover your cursor over the warning icon to view specific information on why the program has called a replicate set invalid To assign replicates with the
40. bars on the relative quantities that reflect the deviation among the replicates The program calculates the error bars on the relative quantities from the error bars on the Cqs which it calculates from the deviation on each fluorescence measurement at each cycle and estimates on the imprecision of the normalizers To add error bars 1 Select the Relative Quantity chart on the Graphical Displays screen 2 Make sure that the program is set to treat replicates collectively See Choose a treatment for replicate wells on page 162 3 Inthe right panel next to Error Bar select On The program add error bars to the chart To remove error bars 1 Select the Relative Quantity chart on the Graphical Displays screen 2 In the right panel next to Error Bar select Off The program removes the error bars from the chart Adjust the graph properties You can adjust certain properties of the Relative Quantities chart e g the scales of the axes the graph title and the background color through the short cut menu and the Graph Properties dialog box See Customize graph properties on page 211 for more information Select the algorithm method The program provides multiple algorithm options for calculating the relative quantities To select an algorithm for the relative quantity calculations 1 Select the Relative Quantity chart on the Graphical Displays screen 2 Inthe right panel click the downward arrowhead above the result t
41. configuration you can save the changes either by overriding the existing definition or by creating a new definition To save changes to a report configuration by overriding the existing definition 1 After loading the saved definition on the Generate Report screen and making changes to the report configuration click the arrow next to the Save icon F to expand the drop down list 2 Click Save The program saves the changes that you made to the report configuration to the loaded definition To save changes to a report configuration by creating a new definition 1 After loading the saved definition on the Generate Report screen and making changes to the report configuration click the arrow next to the Save icon Fi to expand the drop down list 2 Click Save As The Add New Definition dialog box opens 3 In the Definition Name field type a name for the definition or use the name provided 4 Click Add The dialog box closes and the program saves the report configuration to the new definition Agilent AriaMx Real Time PCR Program 271 14 Generating Multiple Experiment Analysis Reports and Exporting Results Export MEA data results to an Excel or text file 272 When working in a project the Export Data screen has tools for exporting numerical data from the experiments to an Excel or text file Data files can include data on the setup of the experiments as well as data from the results of the project To open
42. experiment When you compare the melt data by experiment the program generates a separate graph for each experiment in the project and analyzes the data from each experiment separately To compare by experiment 1 Select the Melt Curve Raw Derivative Curve graph on the Graphical Displays screen 2 In the right panel next to Compare select Experiment Agilent AriaMx Real Time PCR Program Analyzing Multiple Experiment Analysis Project Results 13 Compare raw or derivative melt curves by target When you compare the melt data by target the program generates a separate graph for each target If multiple experiments in the project amplified the same target the program displays the melt curves from these different experiments on the same graph Make sure each unique target has a unique name See Differentiate between targets across experiments on page 244 This view provides a good way to compare how the same target performed in different experiments Note that comparing melt curves by target rather than by experiment does not impact the Tm values that the program calculates for each curve To compare by target 1 Select the Melt Curve Raw Derivative Curve graph on the Graphical Displays screen 2 Inthe right panel next to Compare select Target Consolidate the raw or derivative melt curves When you select to consolidate the melt data the program displays all of the melt curves from all experiments on a single g
43. export reports Enable or disable auto renaming of restored experiments The auto renaming feature is for the automatic renaming of experiments restored to the primary database If you restore an experiment to the primary database but the database already contains an experiment of the same name auto renaming appends a bracketed number to the name of the restored experiment When auto renaming is disabled you cannot restore an experiment if the primary database already contains an experiment of the same name Agilent AriaMx Real Time PCR Program 297 16 Help for the AriaMx ET Electronic Tracking Software To enable auto renaming 1 Open the User Management dialog box to the General Settings tab 2 Under Archive Restore mark the check box 3 Click Close to close the dialog box and apply your changes Auto renaming is now enabled To disable auto renaming 1 Open the User Management dialog box to the General Settings tab 2 Under Archive Restore clear the check box 3 Click Close to close the dialog box and apply your changes Auto renaming is now disabled Manage user accounts The User Settings tab of the User Management dialog box has tools for creating and editing user accounts 298 Agilent AriaMx Real Time PCR Program Help for the AriaMx ET Electronic Tracking Software 16 General Settings User Settings User Settings Export To Excel Print Create User Username Admin Full Name Adnnin
44. for the AriaMx software The AriaMx software contains a help system that provides instructions on setting up running and analyzing experiments and creating multiple experiment projects You can also view video tutorials on the Agilent website that describe how to perform some of the most common tasks in the program Help system From any screen or dialog box click the help icon in the upper right corner of the screen to open the help system The help system automatically opens to the most relevant topic based on where you are in the program You can navigate the help system window from the Contents tab or the Search tab e On the Contents tab use the table of contents on the left side of the window to browse the chapters and topics within the help system e On the Search tab type a search word into the field and click GO to find the help topics that contain the word If you type multiple words into the field the program will list help topics that contain all of the words Use quotes to search for a complete phrase e g comparative quantitation You can also use the Boolean operators AND and OR to find topics that contain more than one search word e g comparative AND quantitation or topics that contain any one of multiple search words e g comparative OR quantitation The help system contains the following chapters e Getting Started with the AriaMx Software Contains help topics to familiarize new users with the
45. have connected to an instrument since last launching the AriaMx program the Login dialog box opens Select your Username from the drop down list type your login password into the Password field and click Login To log in witha different user account right click on the instrument name and click Log off current user You can then log in using the desired user account A message box opens notifying you that you must save the experiment before you can connect to the instrument 5 Click Save in the message box 30 Agilent AriaMx Real Time PCR Program Performing Hardware and Software Tests and HRM Calibrations 3 The Save As dialog box opens 6 Select a folder for the experiment and type a name into the File name field or use the default Click Save 7 Take the prepared qualification test plate over to the instrument and load it into the thermal block 8 On the instrument touchscreen open the primed experiment to the Thermal Profile screen and press Run Experiment The instrument starts running the qualification test experiment 9 Return to the PC program The program directs you to the Raw Data Plots screen where you can monitor the progress of the run See Monitor a run on page 149 10 At the end of the run save the qualification test experiment to your PC see Retrieve run data from the instrument on page 154 for instructions Open the experiment in the AriaMx program on your PC 11 Click Graphical Displa
46. is preferable to method Agilent AriaMx Real Time PCR Program Creating and Setting Up an MEA Project 12 3 below if you observe significant differences in background noise between experiments Method 3 Set identical thresholds in all experiments If you find that the background signal levels are similar between the experiments in your project you can manually set the threshold level for a target to the same value in all experiments The tools used for manually adjusting threshold fluorescence levels in a project are the same as those used for an experiment see Manually adjust threshold fluorescence values on page 177 Agilent AriaMx Real Time PCR Program 239 12 Creating and Setting Up an MEA Project Create an MEA project You can create a new MEA project from the Getting Started screen To open the Getting Started screen At the top of the program window click File gt New or click the icon to open a new tab in the program The new tab opens to the Getting Started screen To create a MEA project 1 2 240 Close all open experiments and projects On the Getting Started screen under New Project click Multiple Experiment Analysis The center of the screen displays the tools for creating a new project Click Add Experiment The Open dialog box opens Browse to the experiment that you want to add to the project Select the experiment press Ctrl to select multiple experiments within the same folder a
47. new database Click OK to close the message box Agilent AriaMx Real Time PCR Program 303 16 Help for the AriaMx ET Electronic Tracking Software Archive experiments To archive an experiment 1 2 Open the Archive dialog box Admin gt Archive Experiments In the Select Archive Database drop down list select the desired archive database In the table mark the check box in the Archive column for the experiments that you want to archive To search for an experiment type a search term into the Search field at the top of the dialog box To archive all experiments mark Select All Experiments to Archive at the bottom of the dialog box Click Archive A message box opens confirming the number of experiments that the program successfully archived In order to archive or restore experiments your PC must be running MSDTC service See the AriaMx Setup and User s Guide for instructions on configuring and starting the MSDIC service Restore experiments Using the Restore dialog box you can take archived experiments out of the archive database and restore them to their original primary database or a different primary database Restoring experiments makes them available for further editing and analysis 304 Agilent AriaMx Real Time PCR Program Help for the AriaMx ET Electronic Tracking Software 16 J Restore Select Archive Database Ariat archive Restore Experiment Name Last Modified a new expt 2 Mar 2
48. of 80 C or higher correspond to the larger PCR products and can usually be assigned as specific DNA product Products displaying melting temperatures of lt 75 C correspond to non specific DNA products such as primer dimers It is important to note however that these populations are not necessarily homogenous and may contain multiple PCR product species Agilent AriaMx Real Time PCR Program 183 10 Viewing Graphical Displays of the Results 184 View data for a single data point Each curve on the graph is the plot for a single target in a single well or replicate set Each data point that makes up the curve is a fluorescence value plotted on the Y axis measured at a particular temperature plotted on the X axis The program connects these data points to draw the raw derivative curves displayed on the graph To view a summary of the data for a single data point on a plot 1 Select the Melt Curve Raw Derivative Curve graph on the Graphical Displays screen 2 Hover your cursor over an individual plot on the graph A tooltip opens displaying the following information e The well ID or replicate number for the plot and the target name e The fluorescence data type for the plot followed by the X and Y coordinates for the data point where your cursor is located e The Tm of each product identified by the algorithm Select a fluorescence data type For the Y axis of the raw derivative curves you can select to use raw or normal
49. of the standard curve to the standard data points plotted The value will always be between 0 and 1 The closer the value is to 1 the better the fit of the line Sigma Sigma is a measurement of the variability standard deviation of the fluorescence measured from all wells and more than one cycle Typically its value is determined from the first few cycles before the PCR reaction starts to affect the measurement The Sigma multiplier is a user defined number that is used to multiply by sigma to create a threshold value for determination of Cq p value The p value refers to the probability that the mean of one set of sample data is different than the mean of another set of sample data The first set of sample data is always the control wells in the analysis selection When replicates are being treated individually the second set of sample data consists of a single well usually an Unknown well When replicates are being treated collectively the second set of sample data consists of all of the replicates If the p value exceeds the user specified confidence level the well dye is given a call whereas if the p value does not exceed the user specified confidence level the well target is given a call Agilent AriaMx Real Time PCR Program Reference Help and Troubleshooting amp Support 17 Confidence Level The user defined confidence level for calls is the statistical probability required before the algorithm will call amplificati
50. option 1 On the Plate Setup screen select all the wells on the plate map that have the same number of wells per replicate set 2 Inthe Properties panel under Replicates select Auto Agilent AriaMx Real Time PCR Program 123 124 Setting Up the Plate Replicates Manual Direction of Assignment Wells per i replicate set B B Horizontal In the Direction of Assignment drop down list specify how the replicate wells are arranged on the plate e Select Horizontal if the replicate reactions will be arranged horizontally in rows e Select Vertical if the replicate reactions will be arranged vertically in columns In the Wells per replicate set field type the number of replicate wells per reaction or click the buttons to enter the desired number The assigned replicate number appears in each selected well If desired make adjustment to the auto assignments in any of the wells of the plate by switching to the Manual option and manually assigning replicate numbers to those wells To manually assign replicates 1 On the Plate Setup screen select a set of wells that are part of the same replicate set Make sure that the selected wells are of the same well type and contain identical targets For instructions on well selection see Select and view wells in the plate map on page 74 In the Properties panel under Replicates select Manual if not already selected In the Assign Replicate Number fie
51. or collectively e On the Analysis Criteria screen click the Replicates toggle button at the bottom of the screen When the button looks like the image on the left the program treats them individually When the button looks like the image on the right the program treats them collectively e se 250 Agilent AriaMx Real Time PCR Program Analyzing Multiple Experiment Analysis Project Results 13 Overview of the Graphical Displays screen for a project The Graphical Displays screen shows the results of the project displayed in a Series of graphs For each graph the screen includes tools for setting certain analysis parameters The screen also includes a result table with configurable columns of data that you can export to an Excel spreadsheet To open the Graphical Displays screen for a project Click Graphical Displays in the Experiment Area panel on the left side of the screen Graphs The exact set of graphs available on the Graphical Displays screen varies depending on the type of experiments in the project but all projects include a graph of the amplification plots and all project in which the experiments have a melt segment include a graph of the raw derivative melt curves note that difference plots are not available in projects See the topics below for detailed information on specific graphs Compare amplification plots in a project on page 256 Compare raw or derivative melt curves in a project on page 258
52. plate to plate variability in fluorescence that is not due to true differences in template concentration between reactions can impact the reliability of such comparisons Although the program was designed to calculate the threshold values in a way that limits the effects of this variability to help ensure the validity of your comparisons you can take steps to reduce variability between experiments see Reducing plate to plate variability below Also consider which method for threshold fluorescence determination is most appropriate for your project see Selecting a method for setting threshold fluorescence levels on page 238 Reducing plate to plate variability Variability comes in many forms e g signal strength noise level background fluorescence and amplification efficiency and may result from many sources e g differences in plates reagents and assay preparation The best way to reduce variability is to use good lab technique and high quality reagents To further limit variability use reagents from the same lots when setting up experiments that you plan to directly compare To monitor for variability in the amplification efficiency of a target run several standard curves on different days The amplification efficiency for a target should be similar from one experiment to the next In a Comparative Quantitation project the AriaMx program allows you to normalize the quantity of a target of interest to a normalizer t
53. port number and click the Delete icon 1i 3 Click OK to save your changes and close the Discovery Port dialog box The instruments from the selected port number are listed in the Instrument Explorer dialog box View information about an instrument For the available instruments listed in the Instrument Explorer dialog box you can view information about the instrument including e The optical modules that are installed on the instrument e The instrument serial number e The IP address for the instrument e The firmware version installed on the instrument 144 Agilent AriaMx Real Time PCR Program Running and Monitoring Experiments 8 To view instrument information 1 In the Instrument Explorer dialog box click the Instrument Information icon next to the desired instrument The Instrument Information dialog box opens Agilent AriaMx Real Time PCR Program 145 8 Running and Monitoring Experiments Start stop or pause a run Starting a run causes the instrument to initiate the thermal profile protocol Start a run To start running an experiment 1 Set up the experiment in the program either using the PC version of the AriaMx program or using the touchscreen program and set up the PCR reactions in the 96 well reaction plate Setting up the experiment in the software requires creating the experiment file setting up the plate and setting up the thermal profile 2 Start the run e If you are working from the PC version of
54. program and provide instructions for getting started e How To Help Provides step by step instructions on how to use the program e Reference Help Contains trademark designations and a glossary of QPCR terms e Troubleshooting and Support Contains troubleshooting suggestions and a directory for contacting a technical support person in your region Agilent AriaMx Real Time PCR Program 21 1 22 Getting Started with the AriaMx Program Video tutorials Visit http www agilent com genomics AriaMxVideoHelp to access video tutorials that describe how to perform a variety of common tasks in the AriaMx program Sample experiments The AriaMx software comes with several sample experiment files that include post run data The sample experiments are saved to the folder C Users Public Documents Agilent AriaMx Sample Experiments during installation of the software The folder includes a sample experiment for each experiment type and subtype You open the sample experiments in the AriaMx software to help familiarize yourself with the experimental setup and graphical displays available for each experiment type Agilent AriaMx Real Time PCR Program 2 Specifying Instrument and Program Settings Update instrument optics 24 Set program preferences 26 2 Agilent Technologies 2 Specifying Instrument and Program Settings Update instrument optics Multiple optics modules are available for use with the AriaMx instrument for
55. program opens a larger schematic of the well that displays the properties assigned to the well Agilent AriaMx Real Time PCR Program 75 76 Setting Up the Plate Zoom in out on the plate map You can zoom in and zoom out on the wells of the plate map using the commands on the plate map short cut menu To zoom in on the wells of the plate map 1 3 Right click anywhere on the plate map The short cut menu opens Click Zoom In The program zooms in on the plate map displaying fewer wells in more detail If desired repeat steps 1 2 to zoom in further To zoom out on the wells of the plate map 1 Right click anywhere on the plate map The short cut menu opens Click Zoom Out The program zooms out on the plate map If desired repeat steps 1 2 to zoom out until the plate map displays all 96 wells View a mini plate map The minimap tool allows you to limit the plate map to specific wells which enables you to view more detailed information on the properties of those wells Agilent AriaMx Real Time PCR Program Setting Up the Plate 6 To view a mini plate map 1 In the Properties panel click the Minimap icon Ge The Minimap box opens This box displays a small schematic of the plate map 2 Minimap 2 Inthe Minimap box click the zoom scroll bar to zoom in on the wells in the minimap The red box outlines the wells to be included in the minimap 2 Minimap 3 Click and drag the red box
56. reading divided by the initial fluorescence reading e The possible options if you selected to display by Cq AR Cq of the baseline corrected raw fluorescence plot ARn Cq of the baseline corrected and normalized fluorescence plot See Select which data collection points to analyze on page 161 for information on specifying which data collection point to use for generating the amplification plots Agilent AriaMx Real Time PCR Program 207 10 Viewing Graphical Displays of the Results Display genotype groups on the graph You can include on the graph colored rectangles that group data points with the same assigned genotype allelic composition You can allow the program to automatically determine the positions of these rectangles or you can manually position them yourself Allele Determination Allele Genotype Allele A Allele E Heterozygous V Mone 2500 2 2000 a 5 1500 ta a 1000 or 500 g g sgg 1000 1500 2000 2500 3000 AR last for HEX To include colored rectangles that are automatically determined by the program 1 Select the Allele Determination graph on the Graphical Displays screen 2 In the right panel next to Genotype Calls select Auto The program adds the rectangles to the graph grouping data points with the same genotype call To include colored rectangles and manually position them 1 Select the Allele Determination graph on the Graphical Displays screen 2 In the right panel next to Ge
57. results 266 View a preview of the report 266 Select report type 266 Generate the report 266 Configure the report 26 7 Create or edit report configuration definitions 2 0 Export MEA data results to an Excel or text file 2 2 Configure the file and export data 2 2 Load a saved data export definition 2 4 Create or edit data export definitions 2 4 fhe Agilent Technologies 14 Generating Multiple Experiment Analysis Reports and Exporting Results Generate report of MEA project results When working in a project the Generate Report screen allows you to set up preview and create a PDF or PowerPoint report for the project The content and format of the report are highly customizable and you can save a report configuration for use with additional projects later on To open the Generate Report screen Click Generate Report in the Experiment Area panel on the left side of the screen View a preview of the report The center of the Generate Report screen displays a page by page preview of what the report will look like with the current configuration settings Above each page is the name of the report item displayed on that page To view all pages of the report preview scroll down To adjust the display size of the pages click the buttons at the bottom of the screen Select report type The report can be a PDF or PowerPoint file To select the report type e In the Report Configuration panel of the Generate Report screen
58. results from multiple experiments of the same type side by side while still treating the experiments independently This capability facilitates comparisons between experiments while still keeping the data separate Agilent AriaMx Real Time PCR Program 235 12 Creating and Setting Up an MEA Project Restrictions You can only use MEA with completed post run experiments that were run on an AriaMx instrument To be grouped into the same project experiments must be of the same experiment type and their thermal profiles need to be similar same type of segments in the same order You can convert experiments to a different type through the Convert Experiment Type command in the File menu The Melt Curve Difference Plots graph is not available for MEA projects even if all the experiments in the project include a high resolution melt segment To view the Difference Plots for an individual experiment in a project open the experiment 236 Agilent AriaMx Real Time PCR Program Creating and Setting Up an MEA Project 12 Guidelines for Comparing Cq Values Across Experiments In a multiple experiment analysis MEA project the program individually calculates a threshold fluorescence value for each target in each experiment and uses these threshold values to derive the quantification cycle Cq values When the data are compared by target you may be comparing the Cq values from one experiment to the values from another experiment Any spurious
59. right click directly on that row 3 Inthe pop up menu that opens click Apply Call to Current Item then click the genotype that you want to assign Homozygous A Homozygous B or Heterozygous If the Results Table is displaying the Call column the call you applied appears in that column To apply a genotype call to multiple Unknown wells or replicates sets 1 Select the Melt Curve Difference Plots graph on the Graphical Displays screen 2 Inthe Results Table add the Check column if not already added a Click the Column Option icon ra above the table to open the Column Options dialog box b Mark Check and click OK 3 In the Results Table locate the wells or replicate sets that you want to assign to the same genotype call For those wells or replicate sets mark the check box in the Check column 4 Right click on the Results Table or on the Difference Plots graph 5 In the pop up menu that opens click Apply Call to All Checked Items then click the genotype that you want to assign Homozygous A Homozygous B or Heterozygous If the Results Table is displaying the Call column then the call you applied appears in that column Clear calls You can remove calls that were manually applied to Unknown wells or replicate sets Agilent AriaMx Real Time PCR Program 193 10 Viewing Graphical Displays of the Results 194 To clear a call for a single Unknown well or replicate set 1 Select the Melt Curve Differen
60. rows By default all rows are initially selected Click the Select All icon at the top of the result table to reselect all rows Data columns You can configure which columns of data are included in the table Click the Column Options icon Pa at the top of the result table to open the Column Options dialog box In the dialog box mark the columns that you want to include in the result table You can freeze one or more columns on the left side of the results table so that as you scroll through the table horizontally the frozen columns are always visible Right click on the header of the right most column that you want to freeze and click Freeze Column To unfreeze right click again and click Unfreeze Column Display options When you have multiple graphs selected for viewing on the Graphical Displays screen you can manually drag and drop the graphs to new positions on the screen using your cursor the Manual Arrange feature Alternatively you can select for the program to automatically arrange the graphs based on the desired number of graphs per screen the Auto Arrange feature Agilent AriaMx Real Time PCR Program Analyzing Multiple Experiment Analysis Project Results 13 To access the options for manually and automatically arranging the graphs click the icon shown below at the bottom of the Graphical Display screen r The following menu opens The options under Manual Arrange and Auto Arrange are described below
61. saved data export definition 2 4 Create or edit data export definitions 2 4 15 How To Examples for Multiple Experiment Analysis Projects 2 77 Example 1 2 8 How to find the initial template quantity of a target in an unknown sample using a standard curve from a separate experiment 2 8 Example2 280 How to normalize target quantities in Unknown and Calibrator wells using a normalizer target from a separate experiment 280 16 Help for the AriaMx ET Electronic Tracking Software 283 Overview of the AriaMx ET software 284 Open an experiment in the AriaMx ET software 286 Open an experiment 286 Import and export experiments in the AriaMx ET software 289 Import experiments into the database 289 Export experiments from the database 289 Lock or log out of the AriaMx ET software 291 Lock the program 291 Log out of the program 291 Change your password in the AriaMx ET software 293 Create a multiple experiment analysis project in the AriaMx ET software 294 Manage users in the AriaMx ET software 295 Set account properties for all users 295 Manage user accounts 298 Archive and restore experiments in the AriaMx ET software 302 Archive experiments 302 Restore experiments 304 Agilent AriaMx Real Time PCR Program 13 View audit trails and system logs in the AriaMx ET software 306 View the audit trail logs 306 View the system logs 309 Add and remove databases in the AriaMx ET software 311 Add an AriaMx ET database 312 Remove an Ar
62. saves a hew snapshot of an experiment each time a user performs a tracked action in the experiment e g changing an analysis setting For each snapshot the logs indicate the user who performed the action the date and time of the action and a description of the action 306 Agilent AriaMx Real Time PCR Program Help for the AriaMx ET Electronic Tracking Software 16 Log Viewer oS r e Search Log Audit Trail Logs System Logs Experiment Name Date Admin Quantitative PCR 10 Fold Example 2 Items Quantitative PCR 10 Fold Example 27 Mar 2014 07 15 09 Quantitative PCR 10 Fold Example 2 Mar 2014 07 16 46 t Quant PCR_SYBR 1 Item t Comparative Quantitation Multiplex Example copy 1 1 Item b f armnsrsathes Cnrantitetion Adoltislesy Pyernolelearirl Ti tarmi Base Experiment Quantitative PCR 10 Fold Example 3 27 2014 7 15 09 AM Time Stamp User Description 27 Mar 2014 07 16 46 Admin Analysis Replicate Off 27 Mar 2014 07 16 46 Admin Analysis Background Based Threshold End Cycle Old value 9 New value 10 Export To Excel Print Open and navigate the audit trail logs The audit trail logs show all available snapshots for each experiment organized first by username then by experiment name To open the audit trail logs and navigate its contents 1 Open the Log Viewer dialog box Admin gt Log Viewer 2 Next to Log select Audit Trail Logs if not already selected The table at the top of the dialog box l
63. that are available for the Standard Curve graph for a single experiment See View the Standard Curve on page 196 for instructions Compare standard curves by experiment When you compare the standard curves by experiment the program generates a separate graph for each experiment in the project and analyzes the data from each experiment separately Note that any unknown samples are only compared to Standard wells that were run on the same plate To compare by experiment 1 Select the Standard Curve graph on the Graphical Displays screen 2 Inthe right panel next to Compare select Experiment Agilent AriaMx Real Time PCR Program Analyzing Multiple Experiment Analysis Project Results 13 Compare standard curves by target When you compare the standard curves by target the program generates a graph for each target in the project Thus all the Cq values for a single target in all Standard and Unknown wells are plotted on the same graph The initial template quantities in the Unknown wells are based on the data from the Standard wells for the same target regardless of whether the Unknown and Standard wells were run on the same plate If Standard samples for the same target were run on multiple experiments in the project and they have all been included in analysis the program plots the Standard wells together on the same curve and calculates initial template quantities of the target in the Unknown wells based on all the collect
64. that you do not want to include If marked the Experiment Name Experiment Type and Run Date appear in the header of the report The Page Number if marked appears in the footer of the report Agilent AriaMx Real Time PCR Program 225 11 226 Generating Reports and Exporting Results Rearrange the pages of the report You can set the order of the items included in the report by dragging and dropping within the report preview To rearrange the pages 1 At the bottom of the Generate Report screen click Rearrange The program adjusts the display of the report preview to show thumbnails of all items included in the report with a number next to each item to indicate its order in the report Note that some items take up more than one page For those items the number of pages is indicated in parentheses after the item name 2 For an item that you want moved to a different order click and drag on the thumbnail Drop the item into the desired order Repeat for any other items you want to rearrange 3 Click Rearrange again The program sets the display of the report preview back to the standard mode with the pages displayed in the new arrangement Create or edit report configuration definitions You can save your definitions for the report configuration as a report configuration definition You can then load the saved definition into other experiments of the same experiment type Save changes to the default report configura
65. the Cycle Range fields under Background Based Threshold Backeround Based Threshold Cycle Range 6 thru B 17 Sigma Multiplier 10 3 In the first field next to Cycle Range type the cycle number that will be the first cycle in the range or click the buttons to adjust the value in the field to the desired cycle number The program automatically recalculates the background noise and adjusts the threshold fluorescence values for all targets that have not been locked Agilent AriaMx Real Time PCR Program Viewing Graphical Displays of the Results 10 4 In the second field type the cycle number that will be the last cycle in the range or click the buttons to adjust the value in the field to the desired cycle number The program automatically recalculates the background noise and adjusts the threshold fluorescence values for all targets that have not been locked To set an accurate threshold you need to set the cycle range to be in the flat baseline range for all plots To change the value of the sigma multiplier 1 Select the Amplification Plots graph on the Graphical Displays screen 2 Inthe right panel click the downward arrowhead above the result table to display the advanced analysis parameter settings 3 Inthe Sigma Multiplier field under Background Based Threshold type the desired value for the sigma multiplier or click the buttons to adjust the value in the field to the desired cycle number The program
66. the Export Data screen Click Export Data in the Experiment Area panel on the left side of the screen Configure the file and export data Before creating the file you can configure the file by selecting which data items to include If desired you can save the configuration as a data export definition which you can later load with a future project see Load a saved data export definition on page 274 Select the items to include in the file To include and exclude item from the file e In the Export Configuration Panel of the Export Data screen under Items mark the check boxes for the items that you want to include in the file Clear the check boxes for any items that you do not want to include The preview of the file in the center of the screen includes only the marked items Above each page is the name of the item To customize the elements of the Plate Setup Thermal Profile Tabular Results Project Notes or Experiment Notes 1 Inthe Export Configuration Panel of the Export Data screen under Items make sure that the check box for the item that you want to customize is marked 2 Click the icon next to the item The Column Options dialog box opens 3 Mark the check boxes for the elements that you want to include in the exported file Clear the check boxes for elements that you do not want to include 4 Click OK in the Column Options dialog box Agilent AriaMx Real Time PCR Program Generating Multiple Expe
67. the Plate Setup screen next to Show select Name The Well Name field becomes available for typing 2 Select all the wells in the plate map that you want to assign to the same well name For instructions on well selection see Select and view wells in the plate map on page 74 You must assign a well to a well type before you can assign it a well name 3 In the Well Name field type the well name for the selected wells Press Enter The Well Name appears at the top of the selected wells 4 Repeat steps 2 3 for all well names that you want to assign Assign sample names After you assign well types you can specify the sample in each well by assigning sample names Each unique template sample included in the experiment can be assigned its own sample name If the two alleles are being amplified in separate wells the sample name is used to associate the wells amplifying Allele A with the wells amplifying Allele B from the same template show Type Name Well Name Sample Name Agilent AriaMx Real Time PCR Program Setting Up the Plate 6 To assign sample names 1 On the Properties panel of the Plate Setup screen next to Show select Sample The Sample Name field becomes available for typing 2 Select all the wells in the plate map that contain the same template sample For instructions on well selection see Select and view wells in the plate map on page 74 3 Inthe Sample Name field type in a na
68. the data point e The fluorescence data type for the plot followed by the X and Y coordinates for the data point e The well name as specified on the Plate Setup screen 206 Agilent AriaMx Real Time PCR Program Viewing Graphical Displays of the Results 10 e The genotype assigned by the program to that target in that well replicate Select data type and fluorescence type to display Each plotted point on the graph represents the coordinates of either the fluorescence values or Cq values for the two targets You can select which type of data fluorescence or Cq you want displayed on the graph To select a data type and fluorescence type 1 Select the Allele Determination graph on the Graphical Displays screen 2 Inthe right panel next to Display by select Fluorescence to plot the fluorescence values or select Cq to plot the Cq values Based on your selection the program displays the options for fluorescence data type 3 Select one of the fluorescence data type options e The possible options if you selected to display by Fluorescence R last the final raw fluorescence reading as measured in the last cycle AR last the final baseline corrected fluorescence reading as measured in the last cycle Rn last the final normalized fluorescence reading as measured in the last cycle ARn last the final baseline corrected normalized fluorescence reading as measured in the last cycle R last R first the final fluorescence
69. the plate map that have the same number of wells per replicate set 2 Inthe Properties panel under Replicates select Auto Replicates Manual Direction of Assignment Wells per replicate set B Horizontal r 3 In the Direction of Assignment drop down list specify how the replicate wells are arranged on the plate Agilent AriaMx Real Time PCR Program Setting Up the Plate 6 e Select Horizontal if the replicate reactions will be arranged horizontally in rows e Select Vertical if the replicate reactions will be arranged vertically in columns 4 In the Wells per replicate set field type the number of replicate wells per reaction or click the buttons to enter the desired number The assigned replicate number appears in each selected well 5 If desired make adjustment to the auto assignments in any of the wells of the plate by switching to the Manual option and manually assigning replicate numbers to those wells To manually assign replicates 1 On the Plate Setup screen select a set of wells that are part of the same replicate set Make sure that the selected wells are of the same well type and contain identical targets For instructions on well selection see Select and view wells in the plate map on page 74 2 Inthe Properties panel under Replicates select Manual if not already selected Replicates GELCTIE Auto Assign Replicate Number 6 Auto Increment wo 3 Inthe
70. the program a Open the Thermal Profile screen Run Status screen or Raw Data Plots screen b Click Run The Instrument Explorer dialog box opens If the open experiment is a post run experiment you will first be prompted to save the experiment with a new name c Locate the instrument that you will be using for the run and click Send Config See Add instruments to your network on page 143 for instructions on searching for and adding instruments e If this is the first time you have connected to an instrument since last launching the AriaMx program the Login dialog box opens Select your Username from the drop down list type your login password into the Password field and click Login To log in with a different user account right click on the instrument name and click Log off current user You can then log in using the desired user account e Ifthe experiment is new you will be prompted to save the experiment before proceeding d Take your reaction plate over to the instrument and load it into the thermal block 146 Agilent AriaMx Real Time PCR Program Running and Monitoring Experiments 8 e On the instrument touchscreen open the primed experiment to the Thermal Profile screen and press Run Experiment The instrument starts running the experiment f Return to the PC program The program directs you to the Run Status screen where you can monitor the progress of the run See Monitor a run on page 149 e I
71. to average results from those wells or treat the wells separately When assigning replicates if you see a flashing red warning Icon in the Properties panel next to Replicates you have an invalid replicate set on the plate To be valid all the wells of a set must be of the same well type and have the same target assignments Hover your cursor over the warning icon to view specific information on why the program has called a replicate set invalid To assign replicates with the Auto option 1 On the Plate Setup screen select all the wells on the plate map that have the same number of wells per replicate set 2 Inthe Properties panel under Replicates select Auto 3 Inthe Direction of Assignment drop down list specify how the replicate wells are arranged on the plate e Select Horizontal if the replicate reactions will be arranged horizontally in rows e Select Vertical if the replicate reactions will be arranged vertically in columns 4 Inthe Wells per replicate set field type the number of replicate wells per reaction or click the buttons to enter the desired number Agilent AriaMx Real Time PCR Program 115 116 Setting Up the Plate The assigned replicate number appears in each selected well If desired make adjustment to the auto assignments in any of the wells of the plate by switching to the Manual option and manually assigning replicate numbers to those wells To manually assign replicates 1 On the Pla
72. you to create a customized plate setup for experiments of the type Allele Discrimination Fluorescence Probe To open the Plate Setup screen When you create a new Allele Discrimination experiment you will automatically be directed to the Plate Setup screen To return to the Plate Setup screen at any time before during or after a run click Plate Setup in the Experiment Area panel on the left side of the screen Assign well types Use the Well Types drop down list to assign well types to all the wells used in the experiment See Well types for Allele Discrimination Fluorescence Probe experiments on page 65 for a description of the available well types To assign well types 1 On the Plate Setup screen select all the wells in the plate map that are of the same type For instructions on well selection see Select and view wells in the plate map on page 74 2 Select a well type from the Well Types drop down list in the Properties panel When the Show setting on the Properties panel is set to Type the well type appears at the top of the selected wells 3 Repeat steps 1 through 2 for all well types to be included in the experiment Agilent AriaMx Real Time PCR Program 111 112 Setting Up the Plate Assign well names After you assign wells to a well type you can if desired assign custom well names Show Type EALER Sample Well Name Sample Name To assign well names 1 On the Properties panel of
73. 014 13 34 41 C Select All Experiments to Restore i Cancel To restore an archived experiment 1 Open the Restore dialog box Admin gt Restore Experiments 2 Inthe Select Archive Database drop down list select the desired archive database 3 Inthe table mark the check box in the Archive column for the experiments that you want to archive To search for an experiment type a search term into the Search field at the top of the dialog box To restore all experiments mark Select All Experiments to Restore at the bottom of the dialog box 4 Click Restore A message box opens confirming the number of experiments that the program successfully restored In order to archive or restore experiments your PC must be running MSDTC service See the AriaMx Setup and User s Guide for instructions on configuring and starting the MSDIC service Agilent AriaMx Real Time PCR Program 305 16 Help for the AriaMx ET Electronic Tracking Software View audit trails and system logs in the AriaMx ET software If you are running the electronic tracking ET version of the AriaMx software and you logged in using an administrator account you have access to the audit trails and system logs that are available in the Log Viewer dialog box To open the Log Viewer dialog box At the top of the program window click Admin gt Log Viewer View the audit trail logs The audit trail logs show all snapshots of an experiment The program
74. 118 3 Set up the thermal profile e Navigate to the Thermal Profile screen Click Thermal Profile in the Experiment Area panel on the left side of the screen e Edit the thermal profile as desired or use the default template thermal profile Agilent AriaMx Real Time PCR Program 43 4 44 Creating Opening an Experiment 4 Run the experiment e From the Thermal Profile screen click Run In the Instrument Explorer dialog box that opens locate the instrument and click Send Config e Load your reaction plate into the instrument s thermal block On the instrument touchscreen open the primed experiment to the Thermal Profile screen and press Run e If desired monitor the progress of the run from your PC 5 Set the analysis criteria e When the run is finished navigate to the Analysis Criteria screen Click Analysis Criteria in the Experiment Area panel on the left side of the screen elect the wells well types and targets to include in the analysis specify the treatment of replicate wells and select the data collection marker to use for analysis If your experiment included a high resolution melt segment HRM assign an HRM calibration plate to the experiment 6 Analyze the data e Navigate to the Graphical Displays screen Click Graphical Displays in the Experiment Area panel on the left side of the screen e View the results of the analysis and customize analysis settings for individual graph
75. 3 Inthe pop up menu that opens click Edit Manual Call Settings The HRM Manual Calling dialog box opens Agilent AriaMx Real Time PCR Program Viewing Graphical Displays of the Results 10 4 For the genotype that you want to assign to a different name type the desired name into the field 5 For the genotype that you want to assign to a different color expand the drop down list to view the color options 6 Click directly on the color that you want to assign 7 Click OK in the dialog box The dialog box closes and color coding in the difference plots is updated Adjust the graph properties You can adjust certain properties of the Melt Curve Difference Plots graph e g the scales of the axes the graph title and the background color through the short cut menu and the Graph Properties dialog box See Customize graph properties on page 211 for more information Agilent AriaMx Real Time PCR Program 195 10 Viewing Graphical Displays of the Results View the Standard Curve 196 For Quantitative PCR experiments Comparative Quantitation experiments and User Defined experiments that include a set of Standard wells the Graphical Displays screen includes a Standard Curve graph This graph is a plot of the Cq Y axis versus the log of the initial template quantity in the Standard wells X axis Each plot is a target in a Standard well or replicate set The graph also plots the Cq values from the Unknown wells The progra
76. Area panel under Project select the experiment for which you want to view the thermal profile The program displays the thermal profile for the experiment in the center of the screen 246 Agilent AriaMx Real Time PCR Program 13 Analyzing Multiple Experiment Analysis Project Results Set analysis criteria for a project 248 Toggle display between one experiment and all experiments 248 Select the wells and well types to include in analysis 248 Select the targets to include in analysis 249 Select which data collection points to analyze 249 Choose a treatment for replicate wells 250 Overview of the Graphical Displays screen fora project 251 Graphs 251 Result table 252 Display options 252 Zooming 254 Compare amplification plots ina project 256 Compare raw or derivative melt curves ina project 258 Compare standard curves ina project 260 Compare Relative Quantity charts ina project 262 Compare Allele Determination graphs in a project 264 Agg Agilent Technologies 13 Analyzing Multiple Experiment Analysis Project Results Set analysis criteria for a project On the Analysis Criteria screen for a project your selections determine the settings that the program uses for data analysis To open the Analysis Criteria screen for a project Click Analysis Criteria in the Experiment Area panel on the left side of the screen The Analysis Criteria screen has an image of the plate map for each experiment in the project The plate maps are based
77. Assign Replicate Number field type in the desired replicate number for the selected wells or click the buttons to enter the desired number The assigned replicate number appears in each selected well To assign replicates using Auto Increment 1 On the Plate Setup screen assign well types as needed for your experiment 2 Inthe Properties panel under Replicates select Manual if not already selected 3 Click Auto Increment Agilent AriaMx Real Time PCR Program 85 6 Setting Up the Plate When you hover your cursor anywhere on the plate map an icon of the number 1 appears next to the cursor 9 4 With the cursor click and drag across the group of wells that you want to assign as replicate number 1 The program assigns the wells to replicate number 1 and the icon next to the cursor changes to a number 2 5 Click and drag across the group of wells that you want to assign as replicate number 2 The program assigns the wells to replicate number 2 and the icon next to the cursor changes to a number 3 6 Continue assigning replicate numbers for the remainder of the plate When finished click Auto Increment to turn off the Auto Increment function Assign quantities to Standard wells In order to generate a standard curve from your data you need to assign the initial template quantity to each Standard well Standard Quantities Select Target crs Starting Amount 0 000e 0 A factor of 1x Units for
78. Auto option 1 On the Plate Setup screen select all the wells on the plate map that have the same number of wells per replicate set 2 Inthe Properties panel under Replicates select Auto Replicates Manual Direction of Assignment Wells per _ m replicate set 6 B Horizontal kd 3 Inthe Direction of Assignment drop down list specify how the replicate wells are arranged on the plate e Select Horizontal if the replicate reactions will be arranged horizontally in rows e Select Vertical if the replicate reactions will be arranged vertically in columns 4 In the Wells per replicate set field type the number of replicate wells per reaction or click the buttons to enter the desired number The assigned replicate number appears in each selected well 108 Agilent AriaMx Real Time PCR Program Setting Up the Plate 6 5 If desired make adjustment to the auto assignments in any of the wells of the plate by switching to the Manual option and manually assigning replicate numbers to those wells To manually assign replicates 1 On the Plate Setup screen select a set of wells that are part of the same replicate set Make sure that the selected wells are of the same well type and contain identical targets For instructions on well selection see Select and view wells in the plate map on page 74 2 Inthe Properties panel under Replicates select Manual if not already selected Replicates CATE Auto Assig
79. BHQ Agilent AriaMx Real Time PCR Program 319 17 320 Reference Help and Troubleshooting amp Support Well Types Unknown Contains a complete reaction mixture including a test template that contains an unknown amount of the specific target Buffer Contains only buffer used to monitor the background fluorescence attributable to the buffer NAC No amplification control contains all reaction components except DNA polymerase NPC No probe control contains all reaction components except the fluorescence labeled probe NTC No template control contains all reaction components except the template nucleic acid Standard Contains a complete reaction mixture including a known concentration of target nucleic acid Used to generate a standard curve which is then used to relate the quantification cycle Cq to initial template quantity in Unknown wells No RT No reverse transcriptase control contains all QRT PCR reaction components except reverse transcriptase Allele A Available in Allele Discrimination experiments and User Defined experiments Contains a complete reaction mixtures with a template sample that is a positive control for homozygous allele A Allele B Available in Allele Discrimination experiments and User Defined experiments Contains a complete reaction mixtures with a template sample that is a positive control for homozygous allele B Mixed Available in Allele Discrimination experiments and User D
80. F to all wells in use in the experiment and displays an R in the wells of the plate map to indicate that the well contains a reference dye Agilent AriaMx Real Time PCR Program 83 6 84 Setting Up the Plate Assign replicates The AriaMx program uses replicate ID numbers to denote technical replicates Technical replicates are QPCR reaction tubes containing identical reaction components and set up using a template from the exact same biological sample source While biological replicates measure the variability in the experimental results due to uncontrolled biological variation from sample to sample technical replicates are used to measure the variability in results that is introduced during the process of experimental setup You can assign replicates using the Manual option or the Auto option When you designate replicate wells on the Plate Setup screen you can set the analysis criteria to average results from those wells or treat the wells separately When assigning replicates if you see a flashing red warning Icon in the Properties panel next to Replicates you have an invalid replicate set on the plate To be valid all the wells of a set must be of the same well type and have the same target assignments Hover your cursor over the warning icon to view specific information on why the program has called a replicate set invalid To assign replicates with the Auto option 1 On the Plate Setup screen select all the wells on
81. HCP to calibrate HRM data from an experiment See If the HCP fails below for information on failed HCPs Save the HCP experiment file to a USB drive so that you can transfer it to your PC where you are running the AriaMx software See Retrieve data using a USB drive for instructions For HCP experiments you Agilent AriaMx Real Time PCR Program Performing Hardware and Software Tests and HRM Calibrations 3 cannot transfer data from the instrument to your PC through the network If the HCP fails If the HCP fails the system s quality check the touchscreen displays a message box at the end of the HCP run notifying you of the failure You will also see a warning icon as shown below if you open the experiment in the AriaMx program on your PC Run Run Status Ay Raw Data Plots Possible causes of a failed plate include pipetting errors during set up of the plate and amplicon degradation If you find your HCP runs repeatedly fail try setting up the plate using the Agilent HRM Calibration Plate kit If problems persist contact Agilent Technical Support see Contact Agilent Technical Support on page 327 Note that you cannot associate a failed HCP with an experiment Agilent AriaMx Real Time PCR Program 37 3 Performing Hardware and Software Tests and HRM Calibrations Run an HRM calibration plate HCP 38 Agilent AriaMx Real Time PCR Program Creating Opening an Experiment Overview of the Getting Started scre
82. Manual Arrange Under Manual Arrange you have two arrangement options Floating arrangement This option allows you to move the graphs to any location on the screen by dragging and dropping them with your cursor Cascade arrangement This option sets the graphs in a cascading arrangement You can move the graphs by dragging and dropping with your cursor Auto Arrange Under Auto Arrange the options determine the number of graphs displayed on the screen 1 2 3 or 4 The image in each icon shows the arrangement of the graphs associated with that option When you select to display more than one graph at a time you can reorder the positions of the graphs by dragging and dropping one graph on top of another with your cursor Agilent AriaMx Real Time PCR Program 253 13 Analyzing Multiple Experiment Analysis Project Results Zooming Within a graph you can zoom in on a particular region of interest Drag your cursor across the region of interest as shown below Amplification Plots wx 3500 3000 j b B B ss Fluorescence AR 500 The program then zooms in on the selected region Amplification Plots a x S Fluorescence AR F J cI 254 Agilent AriaMx Real Time PCR Program Analyzing Multiple Experiment Analysis Project Results 13 To reset the zoom level right click anywhere on the graph and click Reset Zoom For more information on the display options available for the graphs on the Gr
83. Mx Real Time PCR Program Running and Monitoring Experiments 8 Monitor a run The Run Status and Raw Data Plots screens allow you to start a run and to monitor the progress of a run The Run Status screen shows the progression of the run through the thermal profile The Raw Data Plots screen displays the amplification or melt data in each well as it is collected by the instrument in real time To open the Run Status or Raw Data Plots screen Click Run Status or Raw Data Plots in the Experiment Area panel on the left side of the screen Only one computer can monitor the run on a particular instrument at any one time If you are monitoring a run when the run completes the instrument will automatically transfer the run data to the PC unless you logged into the instrument using the guest account If you logged in as guest you must retrieve the data in order to view the results on your PC See Retrieve run data from the instrument on page 154 for more information If you are monitoring a run and someone stops the run from the instrument touchscreen the program opens a message notifying you that the run has been stopped The message will indicate whether or not the user who stopped the run selected to save the partial data from the run If partial data was saved you will need to retrieve it See Retrieve run data from the instrument on page 154 Connect to the running instrument To connect to the instrument that is running th
84. PCR Data Markup Language RDML file format for publication of QPCR data RDML files can contain a large variety of data items This help topic does not describe all the available fields that you can include See www rdml org and the publication in Nucleic Acids Research Nucleic Acids Res 2009 April 37 7 2065 2069 for more information on RDML files When you export data results to an RDML file the program prompts you to select a folder location for the file You can then open the file in an RDML compliant program To configure and export data to an RDML file 1 On the Export Data screen next to File Type select RDML The Experimenter fields appear in the center of the screen 2 Type the First Name and Last Name of the experimenter into the fields in the center of the screen 3 Select and configure the optional fields as desired a Click the icon to expand the options for any particular category b Mark the check boxes for fields that you want to add c Type the information into the new fields 4 Click Export Data The Save As dialog box opens 5 Type a file name into the dialog box and select the folder where you want to save the file Click Save The program creates the file and saves it in the designated folder Load a saved data export definition If you already have a saved data export definition on your system that you want to use for the current experiment you can load that definition from the Export Results scree
85. Project Edit the plate setup of experiments in a project 244 In a multiple experiment analysis project the setup of each plate affects how the program compares the data across experiments To open the Plate Setup screen for a project When you create a new project you are automatically directed to the Plate Setup screen To return to the Plate Setup screen at any time before during or after a run click Plate Setup in the Experiment Area panel on the left side of the screen The Plate Setup screen for a project is very similar to the Plate Setup screen for an experiment See Overview of the Plate Setup screen on page 68 for descriptions of the elements of the Plate Setup screen Select an experiment to edit On the Plate Setup screen you can only edit one plate setup for one experiment at a time To select an experiment for plate setup editing e On the Plate Setup screen in the Experiment Area under Project click directly on the name of the experiment that you want to edit The program displays the plate map for the selected experiment Differentiate between targets across experiments When you compare experiments by target the program analyzes the data and displays results by combining data for each target across all experiments included in the project analysis For this reason if your experiments include multiple targets that were detected using the same dye you need to provide unique target names on each plate i
86. Real Time PCR Program Setting Up the Plate 6 The program assigns the wells to replicate number 1 and the icon next to the cursor changes to a number 2 5 Click and drag across the group of wells that you want to assign as replicate number 2 The program assigns the wells to replicate number 2 and the icon next to the cursor changes to a number 3 6 Continue assigning replicate numbers for the remainder of the plate When finished click Auto Increment to turn off the Auto Increment function Assign quantities to Standard wells In order to generate a standard curve from your data you need to assign the initial template quantity to each Standard well Standard Quantities Select Target Cv Starting Amount 0 000e 0 A factor of en Units for Plate nanograms To assign the standard quantities for a target in the Standard wells 1 On the Plate Setup screen select the Standard wells For instructions on well selection see Select and view wells in the plate map on page 74 2 Inthe Properties panel under Standard Quantities select which target in the selected wells is the standard target i e the target of known quantity in the template 3 Inthe Starting Amount field type in the quantity of the standard target present in the first replicate set of Standard wells You will be asked to specify the units for this amount step 5 This quantity must be either the highest quantity or the lowest quantity in the dilu
87. Replicates are wells that contain identical reaction components repeats You can assign replicates using the Manual option or the Auto option When you designate replicate wells on the Plate Setup screen you can set the analysis criteria to average results from those wells or treat the wells separately When assigning replicates if you see a flashing red warning Icon in the Properties panel next to Replicates you have an invalid replicate set on the plate To be valid all the wells of a set must be of the same well type and have the same target assignments Hover your cursor over the warning icon to view specific information on why the program has called a replicate set invalid To assign replicates with the Auto option 1 On the Plate Setup screen select all the wells on the plate map that have the same number of wells per replicate set 2 Inthe Properties panel under Replicates select Auto Replicates Manual Direction of Assignment Wells per replicate set B g Horizontal 3 Inthe Direction of Assignment drop down list specify how the replicate wells are arranged on the plate e Select Horizontal if the replicate reactions will be arranged horizontally in rows e Select Vertical if the replicate reactions will be arranged vertically in columns 4 Inthe Wells per replicate set field type the number of replicate wells per reaction or click the buttons to enter the desired number The assigned replica
88. Time 55 00 05 Ramp Plateau Temperature C Data collection Segment Number of cycles in each segment Plateau A plateau is a temperature held by the instrument for a specified duration It is represented with a solid horizontal line in the thermal profile with the duration of the plateau displayed directly above the line Agilent AriaMx Real Time PCR Program 129 7 Setting Up the Thermal Profile and the temperature of the plateau displayed directly below the line The valid range for a plateau duration is 1 second to greater than 18 hours The valid temperature range is 25 0 ambient to 99 9 C Ramp A ramp is the transition between two plateaus When you add a new plateau to the thermal profile the program automatically adds ramps leading to and from the new plateau temperature Likewise when you delete a segment or plateau the program removes the associated ramps to connect adjacent plateaus Segment and Number of Cycles A segment is a group of plateaus and the intervening ramps that has been set to cycle at least one time Segments are delineated in the thermal profile by solid vertical lines A cycle is one pass through a segment The cycle number for the segment is displayed at the bottom of the thermal profile The table below lists the types of segments and the maximum number of cycles allowed for each segment type Table Segment types available in AriaMx Segment Description Maximum
89. Up the Plate 6 Show Type Name Well Name Sample Name Biological Replicate ID To assign sample names 1 On the Properties panel of the Plate Setup screen next to Show select Sample The Sample Name field becomes available for typing 2 Select a group of wells that contain the same template sample or a group of wells containing samples that are biological replicates For instructions on well selection see Select and view wells in the plate map on page 74 3 Inthe Sample Name field type in a name for the sample Press Enter The sample name appears at the top the selected wells 4 Repeat steps 2 3 for all samples to be included in the experiment To assign biological replicate ID numbers 1 On the Properties panel of the Plate Setup screen next to Show select Sample The Biological Replicate ID field becomes available for typing 2 Among a group of biological replicate wells select the first sub set of wells that require the same biological replicate ID A separate ID is to be assigned to each biological replicate sample within the group 3 In the Biological Replicate ID field type in a number to be assigned to the selected wells Press Enter The program adds the biological replicate ID number to the end of the sample name at the top of the selected well s 4 Repeat steps 2 3 for the remaining wells that require a biological replicate ID assignment Agilent AriaMx Real Time PCR Program 97 98
90. a list of all experiments in the primary database organized by user see image above Click the experiment name not a snapshot below an experiment to select it Then click Open The dialog box closes and the program opens the experiment to the Plate Setup screen To open the latest snapshot of an experiment from the File menu 1 From the toolbar click File gt Open The Open Experiment dialog box opens with a list of all experiments in the primary database organized by user see image above 2 Click the experiment name not a snapshot below an experiment to select it Then click Open The dialog box closes and the program opens the experiment to the Plate Setup screen Open an earlier snapshot of an experiment You can open a previous snapshot of an experiment using multiple approaches To open an earlier snapshot of an experiment from the Getting Started screen 1 Click the icon to the right of the tabs to open a new tab The new tab opens to the Getting Started screen 2 Under Saved click Browse Database The Browse Database dialog box opens with a list of all experiments in the primary database organized by user see image above Expand the node for the desired experiment to view the snapshots available for the experiment The date and time stamp for the snapshot are listed in the Date column 3 Click a snapshot to select it then click Open The dialog box closes and the program opens the experiment to the Plate
91. a preview of the tabular results based on which columns you select to include To quickly mark all check boxes click Select All To restore the default selections click Restore Defaults 5 Click OK in the Tabular Results Properties dialog box The dialog box closes and the Tabular Results pages displayed in the report preview include your changes To edit the Experiment Notes 1 Inthe Report Configuration Panel of the Generate Report screen under Items make sure that the Experiment Notes check box is marked 2 Click the icon next to the Experiment Notes item The Experiment Notes text box opens 3 In the text box type any notes that you want to add to the experiment and include in the report 4 Click Save to save your changes and close the text box To show analysis settings 1 Inthe Report Configuration Panel of the Generate Report screen next to Show Analysis Settings select Yes In the report the analysis settings for each graph are displayed below the graph Select the contents of the header and footer You can select which pieces of information you want to include in the header and footer on the body pages of report To select the contents of the header and footer e In the Report Configuration Panel of the Generate Report screen under Header amp Footer mark the check boxes for the pieces of information that you want to include in the header or footer Clear the check boxes for any pieces of information
92. able to display the advanced analysis parameter settings Agilent AriaMx Real Time PCR Program 203 10 Viewing Graphical Displays of the Results 204 3 Next to Mode select the method you want to use The options are described briefly below AACgq Livak The AACq method also called the Livak method relies on two assumptions The first assumption is that both the normalizer and the target of interest have amplification efficiencies at or near 100 and that the efficiencies of the two targets do not differ by more than 5 The second assumption is that the amplification efficiency of a target is consistent from one run to the next Any run to run variance is not included in the calculations The AACq method is often referred to as an approximation method and requires a validation step to confirm that efficiencies of your normalizer and target of interest are similar This method is only available if the wells selected on the Analysis Criteria screen include a normalizer target ACq The ACq method is similar to the AACg method in that it relies on the same mathematical assumptions about efficiencies and consistency Unlike the AACq method however the relative quantity in the calibrator sample is not set to 1 0 This method is only available if the wells selected on the Analysis Criteria screen do not include a normalizer target Pfaffl With the Pfaffl method the program takes into account the amplification efficiencies of the no
93. agilent com 901 11 68 90 oweden customercare_sweden agilent com 08 506 4 8960 Switzerland customercare_Switzerland agilent com 0848 60355 60 UK Ireland customercare_UK agilent com 0845 12 9292 All other countries Find local contact information at http www agilent com genomics contactus Agilent AriaMx Real Time PCR Program 327 www agilent com In this book This book gives you instructions for using the AriaMx Real Time PCR software program Agilent Technologies Inc 2014 Revision AQ July 2014 G8830 90000 fhe Agilent Technologies
94. alue in the field 3 Click Close The dialog box closes and the program adjusts the font size according to your selection To change the color assigned to a plot 1 Open the Graph Properties dialog box to the Legend Options tab The table under Plot Legend Properties shows each plot color used in the graph Plot Color column and the name of the plot as described in the legend Legend Label column 2 Locate the plot to which you want to assign a new color and expand the drop down list in the Plot Color column A menu opens displaying standard plot color options 3 Select a color e If the standard menu includes your desired color click directly on it e If you need a color not included on the standard menu click Advanced to view an advanced menu for color selection Use the color picker tools to create a custom color The color menu closes and the new color is displayed in the Plot Color drop down list These options are not available if you selected to hide the legend 4 Click Close Agilent AriaMx Real Time PCR Program 219 10 Viewing Graphical Displays of the Results The dialog box closes and the graph displays the new plot color 220 Agilent AriaMx Real Time PCR Program 11 Generating Reports and Exporting Results Generate report of results 222 View a preview of the report 222 Select report type 222 Generate the report 222 Configure the report 223 Create or edit report configuration definitions 226 Exp
95. ample Name To assign well names 1 On the Properties panel of the Plate Setup screen next to Show select Name The Well Name field becomes available for typing 2 Select all the wells in the plate map that you want to assign to the same well name For instructions on well selection see Select and view wells in the plate map on page 74 You must assign a well to a well type before you can assign it a well name 3 In the Well Name field type the well name for the selected wells Press Enter The Well Name appears at the top of the selected wells 4 Repeat steps 2 3 for all well names that you want to assign Assign sample names After you assign well types you can specify the sample in each well by assigning sample names Each unique template sample included in the experiment can be assigned its own sample name Show Type Name Well Name Sample Name Agilent AriaMx Real Time PCR Program 105 6 Setting Up the Plate To assign sample names 1 On the Properties panel of the Plate Setup screen next to Show select Sample The Sample Name field becomes available for typing 2 Select all the wells in the plate map that contain the same template sample For instructions on well selection see Select and view wells in the plate map on page 74 3 Inthe Sample Name field type in a name for the sample Press Enter The sample name appears at the top of the selected wells 4 Repeat steps 2 3 for
96. aphical Displays screen see Customize graph properties on page 211 Agilent AriaMx Real Time PCR Program 255 13 Analyzing Multiple Experiment Analysis Project Results Compare amplification plots in a project 256 The Amplification Plots graphs on the Graphical Displays screen shows a plot of fluorescence Y axis versus cycles X axis The display of the graphs is dependent on how you select to compare the data To view the Amplification Plots for a project Click Graphical Displays in the Experiment Area panel on the left side of the screen If the Amplification Plots graph is not already displayed click the Amplification Plots icon at the bottom of the screen LT The panel on the right side of the screen has tools for adjusting some of the analysis parameters for the amplification plots These tools are the same as those that are available for the amplification plots for a single experiment See View the Amplification Plots on page 173 for instructions Compare amplification plots by experiment When you compare the amplification data by experiment the program generates a separate Amplification Plots graph for each experiment in the project and analyzes the data from each experiment separately To compare by experiment 1 Select the Amplification Plots graph on the Graphical Displays screen 2 In the right panel next to Compare select Experiment Compare amplification plots by target When you compare t
97. arget in the assay to reduce the effect of these spurious variations that do not reflect true differences in target abundance as a result of experimental treatment Any gene with little to no variance in expression due to the experimental treatment can serve as a normalizer Commonly used examples include housekeeping genes such as GAPDH cyclophilin GUS TFIID or 18S or 28S ribosomal RNA The abundance of the normalizer and the target of interest should be similar In a typical Comparative Quantitation experiment you would set up the wells containing the calibrator sample to run alongside a variety of Agilent AriaMx Real Time PCR Program Selecting an ExperimentType 5 unknown samples to test the effect of some variable on the expression level of one or more genes of interest You can set up the reactions amplify the normalizer target in the same well as the target of interest using multiplexing or set up the reactions to amplify the normalizer and the target of interest in different wells During analysis the program automatically adjusts the levels of the target of interest in both Unknown and Calibrator wells to compensate for differences in the levels of the normalizer The program then compares the normalized value for each unknown sample to the normalized calibrator value and reports the relative quantity for each unknown The expression level of the target of interest in the Calibrator wells is set to 1 0 Establish the ampl
98. arget that was run on a different experiment In this kind of plate to plate comparison you are only comparing the ACq values between experiments rather than directly comparing Cq s thus reducing the effects of variability between experiments For measuring the relative template quantity of a target in an unknown sample however the program requires that the calibrator sample be from the same experiment This requirement avoids Agilent AriaMx Real Time PCR Program 237 12 238 Creating and Setting Up an MEA Project the potential problem of differences in the amplification efficiency between the unknown and calibrator reactions Selecting a method for setting threshold fluorescence levels One of the major concerns when directly comparing the Cq values between experiments is the manner in which the threshold fluorescence levels are set If you consider a single amplification plot raising the threshold will give a later Cq and lowering the threshold will give an earlier Cq So reactions with the same starting concentration of template and identical amplification efficiencies will have different Cq values if you set the thresholds differently Consequently you do not want to vary the way that you set the thresholds between experiments when you are directly comparing the Cq values However variation in the level of background fluorescence between experiments means that it is not always optimal to assign the threshold fluorescence to the
99. as an amplification product if the peak crosses that horizontal line 188 Agilent AriaMx Real Time PCR Program Viewing Graphical Displays of the Results 10 View the Melt Curve Difference Plots The Melt Curve Difference Plots graph on the Graphical Displays screen is typically used for analysis of Allele Discrimination experiments that use a DNA binding dye and a high resolution melt HRM segment to distinguish between alleles The graph displays the difference in fluorescence between two plots during a melt ramp Y axis as a function of temperature X axis The panel on the right side of the screen has tools for adjusting some of the analysis parameters for the melt curves The Melt Curve Difference Plots graph is only available for experiments with e A high resolution melt HRM segment in the thermal profile e An associated HRM calibration plate HCP see Assign an HRM calibration plate on page 163 To view the Melt Curve Difference Plots Click Graphical Displays in the Experiment Area panel on the left side of the screen If the Melt Curve Difference Plots graph is not already displayed click the icon for this graph at the bottom of the screen A If the Difference Plots icon has a warning symbol Ay next to it then you cannot open the Difference Plots until you assign an HRM calibration plate HCP to the experiment See Assign an HRM calibration plate on page 163 for instructions About differen
100. at provide access to menus for making selections on which data you want included in the results Results are displayed on the Graphical Displays screen See the following help topics for instructions on specific tasks for setting the analysis criteria Select the wells and well types to include in analysis on page 159 Select the targets to include in analysis on page 160 Select which data collection points to analyze on page 161 Choose a treatment for replicate wells on page 162 Assign an HRM calibration plate on page 163 only for experiments with HRM segment Note that if you wish to use the default analysis settings you need only select the wells to analyze 158 Agilent AriaMx Real Time PCR Program Setting Analysis Criteria 9 Select the wells and well types to include in analysis From the Analysis Criteria screen you can select which wells and well types to include in the results displayed on the Graphical Displays screen To open the Analysis Criteria screen Click Analysis Criteria in the Experiment Area panel on the left side of the screen Select wells for analysis using the plate map The program s analysis algorithms only use the wells that are selected in the plate map on the Analysis Criteria screen Well selection on the Analysis Criteria screen works that same way as it does on the Plate Setup screen See Select wells in the plate map on page 74 for instructions on well selecti
101. atabases are available to users on your system To open the Add Database dialog box At the top of the program window click Admin gt Add Database To open the Remove Database dialog box At the top of the program window click Admin gt Remove Database Adding a database through the Add Database dialog box does not create a new database it only makes the database available for use by the AriaMx ET program on your PC by adding a reference to the database Similarly removing a database through the Remove Database dialog box does not delete a database it only makes the database unavailable for use by the AriaMx ET program on your PC by removing a reference to the database Agilent AriaMx Real Time PCR Program 311 16 312 Help for the AriaMx ET Electronic Tracking Software Add an AriaMx ET database The Add Database dialog box has tools for adding primary and archive databases J Add Database Connect to Server Server Name Username Password Add Database Reference Database Select Database Cancel Add a primary database Each time a user launches the AriaMx ET program the Login dialog box opens prompting the user to login to an AriaMx ET database Using the Add Database dialog box you can add a database to the list of available databases that appears on the Login dialog box To add a primary database 1 a oi A GW N Open the Add Database dialog box Admin gt Add Database The
102. ated Biological replicates are template samples that were isolated independently but from biologically identical sources see Including biological replicates in comparative quantitation on page 60 for more information on biological replicates Agilent AriaMx Real Time PCR Program 119 6 120 Setting Up the Plate Show Type Name Well Name Sample Name Biological Replicate ID To assign sample names 1 4 On the Properties panel of the Plate Setup screen next to Show select Sample The Sample Name field becomes available for typing Select a group of wells that contain the same template sample or a group of wells containing samples that are biological replicates For instructions on well selection see Select and view wells in the plate map on page 74 In the Sample Name field type in a name for the sample Press Enter The sample name appears at the top the selected wells Repeat steps 2 3 for all samples to be included in the experiment To assign biological replicate ID numbers 1 On the Properties panel of the Plate Setup screen next to Show select Sample The Biological Replicate ID field becomes available for typing Among a group of biological replicate wells select the first sub set of wells that require the same biological replicate ID A separate ID is to be assigned to each biological replicate sample within the group In the Biological Replicate ID field type in a numbe
103. ation efficiencies for the target of interest and normalizer target must be Agilent AriaMx Real Time PCR Program 59 5 60 Selecting an Experiment Type identical in order to allow a direct correction of target levels across samples However if you find from your standard curves that the target of interest and normalizer have different amplification efficiencies the program allows you to compensate for this difference using the settings under Amplification Efficiencies in the Graphical Displays screen To access these settings expand the menu in the panel on the right side of the Graphic Displays screen See Enter the amplification efficiencies for the targets on page 204 for detailed instructions Including biological replicates in comparative quantitation In QPCR biological replicates are template samples that were isolated independently but from biologically identical sources that is sources that are genetically identical are of the same cell type and were treated identically during experimentation For example two samples of cDNA that were isolated from the same tissue source in two different mice that were exposed to identical conditions and have the same genotype would be biological replicates Biological replicates help you determine the level of variability in gene expression for your specific experiment that is due to uncontrolled biological variation from sample to sample When setting up the plate for a Compara
104. ations Run an instrument qualification test 30 Runthe test 30 About the Qualification Test graphical data 31 Perform an Installation Qualification test 33 Run an HRM calibration plate HCP 35 Prepare the plate 35 Runan HCP 36 Ifthe HCP fails 37 Agg Agilent Technologies 3 Performing Hardware and Software Tests and HRM Calibrations Run an instrument qualification test Running a qualification test is one way to test for instrument errors You perform the test using a qualification test plate that must be purchased separately from Agilent part number 5190 7709 The wells of the test plate are pre filled with the QPCR reagent mixture needed to run the test Run the test Running a qualification test requires preparing the plate running the experiment on the AriaMx instrument and checking the results To run a qualification test 1 Prepare the qualification test plate according to the instructions that came with the plate At the top of the program window click Instrument gt Qualification Test A new tab opens for the Qualification Test experiment The experiment opens to the Thermal Profile screen Click Run The Instrument Explorer dialog box opens Locate the instrument that you will be using for the run and click Send Config e If the instrument is not listed see Add instruments to your network on page 143 for instructions on searching for and adding instruments e If this is the first time you
105. automatically recalculates the background noise and adjusts the threshold fluorescence values for all targets Lock or unlock the threshold fluorescence values You can lock the threshold fluorescence value for a given target When locked the threshold fluorescence value is fixed and the functions related to manually adjusting the threshold for that target are disabled Additionally if you make changes to the dataset or adjust any of the algorithm settings the program does not recalculate the threshold fluorescence for that target To lock the threshold fluorescence value for a particular target 1 Select the Amplification Plots graph on the Graphical Displays screen 2 Inthe right panel click the downward arrowhead above the result table to display the advanced analysis parameter settings 3 Under the Threshold Fluorescence click the lock icon next to the target name The image of the lock icon changes from unlocked to locked Agilent AriaMx Real Time PCR Program 181 10 Viewing Graphical Displays of the Results To unlock the threshold fluorescence value for a particular target 1 Select the Amplification Plots graph on the Graphical Displays screen 2 Inthe right panel click the downward arrowhead above the result table to display the advanced analysis parameter settings 3 Under the Threshold Fluorescence click the lock icon next to the target name The image of the lock icon changes from locked to unlo
106. aw or derivative melt curves in a project 258 For projects in which the experiments include a melt segment in the thermal profile the Melt Curve Raw Derivative Curve graph on the Graphical Displays screen displays the fluorescence data collected during the melt segment Y axis as a function of temperature X axis You can use the raw or derivative melt curves to verify that the predominant PCR products are amplicons of the intended target For projects comprised of experiments that include a high resolution melt HRM segment you cannot associate or disassociate an HRM calibration plate with the experiments once the project is created Make the HCP associations in the individual experiments before you create the project Note that the Melt Curve Difference Plots graph is not available in projects To view the Melt Curve Raw Derivative Curve in a project Click Graphical Displays in the Experiment Area panel on the left side of the screen If the Melt Curve Raw Derivative Curve graph is not already displayed click the icon for this graph at the bottom of the screen The panel on the right side of the screen has tools for adjusting some of the analysis parameters for the melt curves These tools are the same as those that are available for the Melt Curve Raw Derivative Curve graph for a single experiment See View the Melt Curve Raw Derivative Curve on page 183 for instructions Compare raw or derivative melt curves by
107. ays screen 2 Hover your cursor over an individual plot on the graph A tooltip opens displaying the following information e The well ID or replicate number for the plot and the dye target name e The fluorescence data type for the plot followed by the X and Y coordinates for the data point where your cursor is located e The Tm of each product identified by the algorithm Manually assign Unknowns to a genotype call Typically you can visually examine the difference plots to determine the genotype of the samples in the Unknown wells Once you have made your determinations you can assign a genotype call to the Unknown wells replicate sets in the program Add the Call column to the Results table Before assigning genotype calls you may want to add the Call column to the Results Table so you can easily see the call assignments The calls you apply appear in this column To add the Call column 1 Click the Column Option icon Fa above the table to open the Column Options dialog box 2 In the dialog box mark Call 3 Click OK The dialog box closes and the Call column appears in the Results Table Agilent AriaMx Real Time PCR Program Viewing Graphical Displays of the Results 10 Apply calls To apply a genotype call to a single Unknown well or replicate set 1 Select the Melt Curve Difference Plots graph on the Graphical Displays screen 2 Inthe Results Table locate the row for the well or replicate set of interest and
108. base 1 From the toolbar click File gt Import From File System The Open dialog box opens 2 Browse to the folder location of the experiment Select the experiment and click Open The dialog box closes The program creates a copy of the experiment and saves it to the primary database You can now open the experiment as described in Open an experiment in the AriaMx ET software on page 286 Export experiments from the database Exporting an experiment creates an uncontrolled copy of the experiment You may need to export an experiment in order to make it available for viewing to users outside your network Agilent AriaMx Real Time PCR Program 289 16 Help for the AriaMx ET Electronic Tracking Software To export an experiment from the database to a folder 1 From the toolbar click File gt Export To File System The Export Experiments dialog box opens listing all experiments in the primary database organized by user F 2 Export Experiments Ss te ls Select Experiment To Export Search q Experiment Hame a ate Username gt Admin Experiment name gt Quantitative PCR 10 Fold Example Snapsh ots of the Quantitative PCR 10 Fold Example 27 Mar 2014 Quantitative PCR 10 Fold Example 27 Mar 2018 experiment Cither Users Export To Folder Browse Cancel 2 Mark the check box for the experiment or snapshot that you want to export Marking the experiment will export all snapshots of the
109. box next to the well type name You can remark the check box at any time to add the well type back to the raw data plots Change the scale or orientation of the axes By default the X and Y axes of the graph on the right side of the Raw Data Plots screen are oriented in ascending order and the ranges of the axes adjust automatically based on the plots being displayed To change the orientation of the X or Y axis 1 Right click anywhere on the large graph of the Raw Data Plots screen A short cut menu opens 2 Click Axis Options gt Reverse Orientation in X Axis or Axis Options gt Reverse Orientation of Y Axis The program reverse the orientation of the selected axis To change the scale of the X or Y axis 1 Right click anywhere on the large graph of the Raw Data Plots screen A short cut menu opens 2 Click Axis Options gt Customize Scale The Graph Properties dialog box opens to the Axis Options tab Agilent AriaMx Real Time PCR Program Running and Monitoring Experiments 8 3 Under the heading for the desired axis change the Autoscale setting to Manual 4 Inthe Min and Max fields type the desired minimum and maximum values for the scale of the axis 5 Click Close to save your changes and close the Graph Properties dialog box The program sets the scale of the axis to the new values Stop monitoring a run When you stop monitoring a run the instrument continues running the experiment and the run becomes availabl
110. brator samples See Create an MEA project on page 240 for detailed instructions Step 2 Ensure the correct target is designated as the normalizer View the plate setup for the experiment that includes the normalizer target Make sure that the wells in which the normalizer was amplified have the correct dye assigned in the Normalizer Dye drop down list Ensure that no other targets are also designated as a normalizer in either experiment in the project Agilent AriaMx Real Time PCR Program How To Examples for Multiple Experiment Analysis Projects 15 Step 3 Ensure the sample name assignments are correct To normalize data for a target of interest to a normalizer target that was amplified in a different experiment be sure to assign the same sample name to the wells on both plates that contain the same template sample You can assign sample names from the Plate Setup screen See Assign sample names and biological replicates on page 96 Step 4 Select the analysis criteria Once you have ensured that the normalizer and sample name assignments are correct you can set the analysis criteria for the project on the Analysis Criteria screen Instructions on the controls and settings in this screen an be found in Set analysis criteria for a project on page 248 Step 5 View the relative quantity calculations To view the relative quantities in the Unknown and Calibrator wells open the Graphical Displays screen If the
111. ce Plots graph on the Graphical Displays screen In the Results Table locate the row for the well or replicate set of interest and right click directly on that row In the pop up menu that opens click Apply Call to Current Item gt Clear Call If the Results Table is displaying the Call column the call no longer appears in that column To clear a call from multiple Unknown wells or replicate sets 1 Select the Melt Curve Difference Plots graph on the Graphical Displays screen In the Results Table add the Check column if not already added a Click the Column Option icon above the table to open the Column Options dialog box b Mark Check and click OK c In the Results Table locate the wells or replicate sets for which you want to clear the manually applied genotype call For those wells or replicate sets mark the check box in the Check column d Right click on the Results Table or on the Difference Plots graph e In the pop up menu that opens click Clear All Checked Items If the Results Table is displaying the Call column the calls no longer appears in that column Edit manual call settings From the HRM Manual Calling dialog box you can edit the name and color that is used to for each genotype in the Difference Plots graph and the result table 1 Select the Melt Curve Difference Plots graph on the Graphical Displays screen Right click on the Results Table or on the Difference Plots graph
112. ce plots Difference plots are a useful way of viewing the results of an Allele Discrimination experiment that uses high resolution melt HRM analysis for SNP single nucleotide polymorphism genotyping The values plotted on the Y axis are the difference in fluorescence between a target in one well or replicate set and a control target from a designated well replicate set Because the plots display the difference in fluorescence you can detect even slight differences between two plots Consequently this graph allows you to use HRM analysis to distinguish between product populations that differ in sequence by as little as a single nucleotide Agilent AriaMx Real Time PCR Program 189 10 190 Viewing Graphical Displays of the Results The equation for determining the Y axis value at each temperature is Diff T R T compR T where R T is the fluorescence value for the target at temperature T and compR T is the value for the control target at temperature T Note that the fluorescence value can be any fluorescence term R Rn R T or Rn T The figure below displays a difference plot for a class 4 SNP A gt T Note the distinctly different plot shape for each genotype group Melt Curve Difference Plots 0 10 Homozygous mutant samples _ 0 05 pee a a Am m ES D Ei N E Lin k j 0 05 Homozygous Hetprozyyotts wild type samples samples New 0 10 73 0 74 0 75 0 76 0 Fi ra g
113. cence Probe License Agilent Technologies Reserved 2014 40 Contact Support Agilent AriaMx Real Time PCR Program Creating Opening an Experiment 4 Tools available on the Getting Started screen The content in the center of the screen changes depending on which option is selected in the panel on the left These options are described in the table below a ma Experiment Types Click this option to create a new experiment based on the desired experiment type The center of the screen displays the experiment types for selection My Templates Click this option to create a new experiment from a template The center of the screen displays the template files in the Experiments Templates folder that is created during program installation Multiple Experiment Click this option to create a new project for analyzing and comparing multiple Analysis post run experiments The center of the screen displays tools for adding experiments to the new project Recently Opened Click this option to open an existing experiment or project that you recently accessed The center of the screen displays a list of experiments that you have recently opened Browse Click this option to open an existing experiment or project by browsing to a desired folder Links at the bottom of the screen License Click this link to open a message box about licensing In the message box click License Agreement to view the full text of the AriaMx software license agreemen
114. cked 182 Agilent AriaMx Real Time PCR Program Viewing Graphical Displays of the Results 10 View the Melt Curve Raw Derivative Curve For experiments that include a melt segment in the thermal profile the Melt Curve Raw Derivative Curve graph on the Graphical Displays screen displays the fluorescence data collected during the melt segment Y axis as a function of temperature X axis The panel on the right side of the screen has tools for adjusting some of the analysis parameters for the melt curves To view the Melt Curve Raw Derivative Curve Click Graphical Displays in the Experiment Area panel on the left side of the screen If the Melt Curve Raw Derivative Curve graph is not already displayed click the icon for this graph at the bottom of the screen A About raw derivative curves When using SYBR Green or EvaGreen dye for detection the raw derivative curves are used to verify that the predominant PCR products are amplicons of the intended target The graph plots fluorescence or the first derivative of the fluorescence versus temperature The temperatures plotted on the graph are typically from a melt segment ramp that included a data collection point Plotting results based on the first derivative multiplied by 1 fluorescence term R T or Rn T allows you to view the amplification products as peaks on the graph with each peak centered on the melting temperature Tm for that product Products with a Tm
115. click the icon to open a new tab in the program The new tab opens to the Getting Started screen Create an experiment based on experiment type When you create a new experiment based on experiment type your selection of the experiment type determines some of the setup options and analysis outputs The thermal profile of the new experiment is set to the default for the chosen experiment type The plate setup of the experiment is blank but the available well types and other well configuration tools on the Plate Setup screen are specific to the experiment type To create an experiment based on experiment type 1 Open the Getting Started screen 2 Under New Experiment click Experiment Types The center of the screen displays all the available experiment types for the AriaMx See Overview of Experiment Types on page 54 Click the desired experiment type to select it 4 In the Experiment Name field type a name for the new experiment then click Create The program creates the new experiment and opens the experiment to the Plate Setup screen At step 3 you can double click the desired experiment type to select it with the default experiment name The program creates the new experiment and opens the experiment to the Plate Setup screen or to the Thermal Profile screen for User Defined experiments Agilent AriaMx Real Time PCR Program Creating Opening an Experiment 4 Create an experiment from a template When you create
116. command has a check mark next to it that indicates that the axis Is oriented in the reverse direction The absence of a check mark indicates that the axis is oriented in the forward ascending direction These two commands are part of the Axis Options sub menu Click directly on one of these commands to turn on or off the AutoScale functionality When turned on this functionality causes the program to automatically set the range of the axis based on the data points present on the graph When a command has a check mark next to it that indicates that AutoScale is on The absence of a check mark indicates that AutoScale Is off This command which is part of the Axis Options sub menu opens the Graph Properties dialog box to the Axis Options tab You can customize the scale and range of the axes using the tools on this tab See Customize the graph axes on page 216 Agilent AriaMx Real Time PCR Program Apply Call to All Checked ltems gt Homozygous A Homozygous B Heterozygous Clear Call Clear Checked Items Edit Manual Call Settings Axis Options gt Enable X Axis Log Scale Enable Y Axis Log Scale Axis Options gt Reverse Orientation in X Axis Reverse Orientation in Y Axis Axis Options gt Enable X Axis AutoScale Enable Y Axis AutoScale Axis Options gt Customize Scale Viewing Graphical Displays of the Results 10 These commands are used for manual genotype calling in the Melt Curve Difference Pl
117. cons The top right corner of the program window has 3 icons for quickly accessing the Home screen viewing any notifications from the program and opening the AriaMx help system gt Click this icon to open the Home screen Click the icon again to close the Home screen and return to previous screen Click this icon to open a text box displaying any notifications from the program When you have unread notifications waiting the icon is dark blue Click this icon to open the program s help system to the topic that pertains to the currently displayed screen Getting Started screen When you open a new tab in the program from the File menu or the icon it opens to the Getting Started screen From this screen you can create a new experiment from scratch or from a template create a new multiple experiment analysis project or open an existing experiment or project See Overview of the Getting Started screen on page 40 for more information Experiment Notes Project Notes The Experiment Notes icon or Project Notes icons appears in the lower left corner of the screen whenever an experiment or project is open Clicking the icon opens the Experiment Notes or Project Notes text box Use this text box to type your own notes pertaining to the particular experiment or project Click Save to save your notes for later reference Agilent AriaMx Real Time PCR Program Getting Started with the AriaMx Program 1 Help access
118. crimination Experiment Type Fluorescence Probe In this experiment type two fluorescent probes labeled with two spectrally distinct dyes are used to discriminate between the two alleles and subsequently determine the genotype of a sample For example if the program detects amplification in an unknown DNA sample for the dye identifying the wild type allele but not for the dye identifying a mutant allele the program designates the sample as wild type homozygous Allele Discrimination Experiment Type DNA Binding Dye Including HRM When using this experiment type you set up the experiment to amplify all alleles in the same well using the same set of primers and the program detects all alleles using the same double stranded DNA binding dye such as SYBR Green or EvaGreen dye The thermal profile includes Agilent AriaMx Real Time PCR Program Reference Help and Troubleshooting amp Support 17 a high resolution melt HRM segment so that melt curves of the targets can be generated Even DNA amplicons that differ in sequence by only a single nucleotide will yield slightly different melt curves An Allele Discrimination experiment that uses HRM analysis for allele discrimination should include positive control samples for each base pair possibility at the SNP location homozygous as well as heterozygous positive control samples Reference Dye Passive dye used for normalization of the fluorescence signal of the reporter dye or fluorophore T
119. ct to the Plate Setup screen You may be prompted to save any open experiments and or close any open tabs Agilent AriaMx Real Time PCR Program 241 12 Creating and Setting Up an MEA Project Select experiments for a project 242 You can add or remove experiments from an existing project You can also include and exclude specific experiments from the project analysis Add or remove experiments from the project When you first create a project the program prompts you to select the experiments that you want to add to the project see Create an MEA project on page 240 You can also add experiments after the project is created as well as delete experiments from the project You can add up to 8 experiments in a single project To add experiment to an existing project 1 2 Open the project to any screen In the Experiment Area next to Project click the icon The Experiments Selection Window opens This window contains a table listing the experiments that are currently in the project Click Add Experiment The Open dialog box opens Browse to the folder that contains the experiment you want to add Select the experiment and click Open To select multiple experiments in a single folder press Ctrl as you select the experiments The Open dialog box closes and the selected experiment appears in the table on the Experiments Selection Window Repeat steps 3 4 for all experiments that you want to include in the exper
120. d and met Agilent AriaMx Real Time PCR Program 1 Getting Started with the AriaMx Program 15 The AriaMx program 16 Getting Started with the AriaMx Software 17 Introduction to the AriaMx software 17 Overview of the user interface 19 Home screen 19 Menu toolbar 19 Tabs 19 Left and right panels 19 Home Notifications Help icons 20 Getting Started screen 20 Experiment Notes Project Notes 20 Help access for the AriaMx software 21 Help system 21 Video tutorials 22 Sample experiments 22 2 Specifying Instrument and Program Settings 23 Update instrument optics 24 Set program preferences 26 3 Performing Hardware and Software Tests and HRM Calibrations Run an instrument qualification test 30 Runthetest 30 About the Qualification Test graphical data 31 Perform an Installation Qualification test 33 Run an HRM calibration plate HCP 35 Prepare the plate 35 Runan HCP 36 ifthe HCP fails 37 Agilent AriaMx Real Time PCR Program 29 4 Creating Opening an Experiment 39 Overview of the Getting Started screen 40 Tools available on the Getting Started screen 41 About the AriaMx file types 42 Quick Start Protocol 43 How to create set up run analyze and generate reports for an experiment Create a new experiment 46 Create an experiment based on experiment type 46 Create an experiment from a template 47 Open an existing experiment 48 Save a copy of an existing experiment 49 Create a template from an existing exper
121. data plots 1 On the Raw Data Plots screen hover your cursor over the Data Collection Marker icon at the bottom of the screen A window opens displaying the thermal profile with data collection markers 2 Click the data collection marker that you want to use for the raw data plots The window closes and the program uses the selected data collection marker in the raw data plots Select which targets to include in the plots By default the raw data for all targets in use on the plate are included in the plots To select specific targets to display in the raw data plots 1 On the Raw Data Plots screen hover your cursor over the Display Targets icon at the bottom of the screen O A window opens showing all targets in use on the plate Agilent AriaMx Real Time PCR Program 151 152 Running and Monitoring Experiments 2 For any targets that you do not want displayed clear the check box next to the target name You can remark the check box at any time to add the target back to the raw data plots Select which well types to include By default the raw data plots are displayed for all well types in use on the plate To show the raw data for only specific well types 1 On the Raw Data Plots screen hover your cursor over the Display Well Types icon at the bottom of the screen ooo eo ooo A window opens showing all well types in use on the plate 2 For any well types that you do not want to include clear the check
122. de of the screen has tools for adjusting some of the analysis parameters for the amplification plots To view the Amplification Plots Click Graphical Displays in the Experiment Area panel on the left side of the screen If the Amplification Plots graph is not already displayed click the Amplification Plots icon at the bottom of the screen LT View data for a single data point Each curve on the amplification plots is the plot for a single target ina single well or replicate set Each data point that makes up the curve is a fluorescence value plotted on the Y axis measured at a particular cycle number plotted on the X axis The program connects these data points to draw the curves displayed on the graph To view a summary of the data for a single data point on a plot 1 Select the Amplification Plots graph on the Graphical Displays screen 2 Hover your cursor over an individual plot on the graph A tooltip opens displaying the following information e Well ID or replicate number for the plot e Target e Well type e Fluorescence data type for the plot followed by the X and Y coordinates for the data point where your cursor is located e If replicates are being treated individually Baseline range starting cycle number ending cycle number used to calculate baseline corrected fluorescence values for that plot Agilent AriaMx Real Time PCR Program 173 10 Viewing Graphical Displays of the Results Select a fluorescence data t
123. default definition loaded on the Export Data screen make your desired changes to the settings 2 Inthe Export Configuration Panel next to Definition expand the drop down list and click Add New The Add New Definition dialog box opens 3 In the Definition Name field type a name for the definition or use the name provided 4 Make sure the Use Current Settings check box is marked Click Add Agilent AriaMx Real Time PCR Program 231 11 Generating Reports and Exporting Results The dialog box closes and the program saves the data export configuration to the new definition Save changes to a custom data export definition If you loaded a saved definition other than the default definition and then made changes to the data export settings you can save the changes either by overriding the existing definition or by creating a new definition To save changes to the data export settings by overriding the existing definition 1 After loading the saved definition on the Export Data screen and making changes to the settings click the arrow next to the Save icon to expand the drop down list 2 Click Save The program saves the changes that you made to the settings to the loaded definition To save changes to the data export settings by creating a new definition 1 After loading the saved definition on the Export Data screen and making changes to the settings click the arrow next to the Save icon A to expand the drop d
124. detection of different dye spectra In the Supported Optical Configuration dialog box you can view the optics modules and their currently supported dyes Periodically Agilent may add new supported dyes and optical modules to the AriaMx Real Time PCR system and make a new optics configuration file available to you You can use the Supported Optical Configuration dialog box to load that new file To open the Supported Optical Configuration dialog box At the top of the program window click Instrument gt Optical Configuration Vij Supported Optical Configuration Sal List of supported optical modules and dyes FAM HEX JOE cY3 OSYBR QUHEX Ocy3 OFAM QJOE Ovic ROX OTEXASRD OROX 24 Agilent AriaMx Real Time PCR Program Specifying Instrument and Program Settings To update the optical configuration file 1 Open the Supported Optical Configuration dialog box 2 Click Import Optical Configuration The Open dialog box opens 3 Browse to the folder of the configuration file that you received from Agilent Select the file and click Open The Supported Optical Configuration dialog box is updated with the new configuration Agilent AriaMx Real Time PCR Program 2 25 2 Specifying Instrument and Program Settings Set program preferences The Preferences dialog box is used for setting your preference on the default file name configuration To open the Preferences dialog box At the top of the program window click File
125. dit Grid Line Color Edit Background Color Edit Graph Title 214 This command is part of the Legend Options sub menu Click directly on Show Legend to show or hide the graph legend When the command has a check mark next to it that indicates that the legend is currently being displayed The absence of a check mark indicates that the legend is currently hidden This set of four options is part of the Legend Options sub menu and is only available when the Show Legend command is marked i e the legend is shown on the graph Use these options to set the location of the legend within the graph This command which is part of the Legend Options sub menu opens the Graph Properties dialog box to the Legend Options tab You can customize the plot colors and legend font size using the tools on this tab See Customize the graph legend on page 218 These four commands are part of the Grid Options sub menu Use these commands to set the display of grid lines on the graph When a command has a check mark next to it that indicates that it is turned on The absence of a check mark indicates that it is turned off When the Show Both Axis Grid Lines command is turned on the graph displays horizontal and vertical grid lines When the Hide Grid Lines command is turned on the graph does not display any grid When the Show X Axis Grid Lines or Show Y Axis Grid Lines command ts turned on only vertical or horizontal grid lines are displayed respecti
126. ds a check mark to the selected well or replicate set Once you check items use the Apply Call to All Checked Items command to assign a genotype call Alternatively you can add a check mark to a well or replicate set using the Check column in the Results Table Apply Call to Current Item gt These commands are used for manual genotype calling in the Melt Curve Difference Plots graphs They are only available when you right click on an Unknown well or replicate set in the Results Table Select one of the genotypes Homozygous A Homozygous B or Heterozygous to assign a Call or select Clear Clear Call Call to remove an existing call Homozygous A Homozygous B Heterozygous Agilent AriaMx Real Time PCR Program 211 10 Viewing Graphical Displays of the Results Apply Call to All Checked ltems gt Homozygous A Homozygous B Heterozygous Clear Call Clear Checked Items Edit Manual Call Settings Axis Options gt Enable X Axis Log Scale Enable Y Axis Log Scale Axis Options gt Reverse Orientation in X Axis Reverse Orientation in Y Axis Axis Options gt Enable X Axis AutoScale Enable Y Axis AutoScale Axis Options gt Customize Scale 212 These commands are used for manual genotype calling in the Melt Curve Difference Plots graphs If you added a check mark to specific wells or replicate sets using the Check Item command or the Check column in the Results Table use these commands to assign the c
127. e Agilent AriaMx Real Time PCR Program 69 6 70 Setting Up the Plate Discrimination and User Defined experiment types also allow you to assign sample names to designate the template sample used in each well The program displays the sample name at the top of each well when the Show setting is set to Sample Target information All experiment types require at a minimum that you designate the dyes being used for detection in the wells You may also assign a name to the target being detected by each dye If the same dye will be used to amplify multiple targets in the experiment assigning a unique name to each target allows the program to distinguish between these targets during analysis Replicate number If some of the wells on the plate are technical replicates of each other i e they have the exact same reaction components you can designate the replicate wells during plate setup by assigning them the same replicate number During analysis you can choose to have the results from replicate wells averaged together at each cycle or you can treat replicates separately to monitor for well to well variation among identical reactions Invalid Sets When assigning replicates if you see a flashing red warning icon in the Properties panel next to Replicates you have an invalid replicate set on the plate To be valid all the wells of a set must be of the same well type and have the same target assignments Hover your cursor over the
128. e Search field at the top of the dialog box View transaction logs for an archive database The Transaction Log dialog box can display all the experiments that have been restored from an archive database to the primary database To view the log of restoration transactions 1 Open the Transaction Logs dialog box Admin gt Transaction Log 2 Next to Database select Archive 3 Inthe Select Archive Database drop down list select the archive database for which you want to view the logs The table lists contains the following columns e Experiment Name experiments that have been archived restored e Archived From Database primary database from which the experiment was archived e Archived On date and time that the experiment was archived e Restored To Database primary database to which the experiment was restored e Restored On date and time that the experiment was restored To help locate a particular experiment type a search term into the Search field at the top of the dialog box 316 Agilent AriaMx Real Time PCR Program 17 Reference Help and Troubleshooting amp Support QPCR Glossary 318 Trademarks 324 Troubleshooting and Support 325 Troubleshooting Guide 325 Contact Agilent Technical Support 327 Agg Agilent Technologies 17 Reference Help and Troubleshooting amp Support OPCR Glossary 318 Experiment Type and QPCR Detection Chemistry Terms Quantitative PCR Experiment Type Experiments of this t
129. e that is known to be homozygous for allele A Home Allele B Contains a complete reaction mixture with a positive control template that is known to be homozygous for allele B Hetero Contains a complete reaction mixture with a heterozygous positive control template that is known to include both allele A and allele B Agilent AriaMx Real Time PCR Program 63 5 Selecting an Experiment Type The Allele Discrimination Fluorescence Probe Experiment Type 64 The Allele Discrimination experiment type is used to discriminate between two alleles of a gene in a genomic DNA or cDNA sample An Allele Discrimination experiment can be performed two different ways using fluorescent probes as described below or using a double stranded DNA binding dye Using the probe method for allele discrimination two fluorescent probes labeled with two spectrally distinct dyes are used to discriminate between the two alleles and subsequently determine the genotype of a sample For example if the program detects amplification in an unknown DNA sample for the dye identifying the wild type allele but not for the dye identifying a mutant allele the program designates the sample as wild type homozygous If the program detects amplification in an unknown DNA sample for the dye identifying the mutant allele but not for the dye identifying the wild type allele the program designates the sample as mutant homozygous If the program detects amplification for bot
130. e Probe experiments Well Well Type Description be a complete reaction mixture including a test template that contains an unknown amount of the specific target No template control contains all reaction components except the template nucleic acid This control is useful for detecting amplicon contamination Homo Allele A Contains a complete reaction mixture with a positive control template that is known to be homozygous for allele A Home Allele B Contains a complete reaction mixture with a positive control template that is known to be homozygous for allele B Hetero Contains a complete reaction mixture with a heterozygous positive control template that is known to include both allele A and allele B Agilent AriaMx Real Time PCR Program 65 5 Selecting an Experiment Type The User Defined Experiment Type The User Defined experiment type provides the greatest flexibility in setup and analysis of an experiment All the well types and other plate setup options that are available across the other three experiment types are available on the Plate Setup screen in a User Defined experiment Similarly on the Thermal Profile screen you can add any type of segment to the thermal profile and on the Graphical Displays screen you can view the results for any of the experiment type specific graphs 66 Agilent AriaMx Real Time PCR Program Setting Up the Plate Overview of the Plate Setup screen 68 The plate map 69 Wellty
131. e Quantity icon at the bottom of the screen The panel on the right side of the screen has tools for adjusting some of the analysis parameters for the relative quantities These tools are the same as those that are available for the Relative Quantity chart for a single experiment See View the Relative Quantity on page 201 for instructions Compare relative quantities by experiment When you compare the relative quantities by experiment the program generates a separate chart for each experiment in the project and analyzes the data from each experiment separately All targets except the normalizer that were run in both an Unknown well and a Calibrator well on the same experiment are included in the chart for that experiment The program normalizes the data for a target of interest to the normalizer target that was run on the same experiment To compare by target 1 Select the Relative Quantity chart on the Graphical Displays screen 2 Inthe right panel next to Compare select Target Agilent AriaMx Real Time PCR Program Analyzing Multiple Experiment Analysis Project Results 13 Compare relative quantities by target When you compare the relative quantities by target the program generates a graph for each target of interest that was run in both an Unknown well and a Calibrator well on the same experiment the program does not compare Unknown wells to Calibrator wells from a different experiment In a by target comparison
132. e available segment types New Segment RT UDG DNA Hot Start Amp Step Amp 3 Step 1 Cycle Agilent AriaMx Real Time PCR Program 133 7 134 Setting Up the Thermal Profile 3 Inthe placeholder click the type of segment that you want to add The program adds the new segment to the thermal profile Refer to the table in Segment and Number of Cycles for a description of each available segment type 4 Edit the plateaus or number of cycles for the segment as desired To remove a segment 1 On the display hover your cursor over the segment that you want to remove An X appears at the top of the segment to the right of the segment name Click the X The program deletes the segment from the thermal profile To move a segment 1 Click and drag the segment that you want to move to the left or right The purple line indicates where in the thermal profile you have dragged the segment When the purple line is in the desired location release the left mouse button to drop the segment into that position To change the number of steps in an amplification segment 1 On the display right click on the amplification segment A short cut menu opens If you want to change from a 3 step amplification to a 2 step amplification click Change to Amp 2 Step If you want to change from a 2 step amplification to a 3 step amplification click Change to Amp 3 Step The program adjusts the numb
133. e experiment that you want to monitor 1 At the top of the program window click Instrument gt Instrument Explorer 2 Locate the instrument and click Monitor Run If the instrument is not listed see Add instruments to your network on page 143 for instructions on searching for and adding instruments If this is the first time you have connected to an instrument since last launching the AriaMx program the Login dialog box opens Select your Username from the drop down list type your login password into the Password field and click Login Agilent AriaMx Real Time PCR Program 149 150 Running and Monitoring Experiments The program directs you to the Run Status screen Monitor a run by viewing its progress through the thermal profile The Run Status screen allows you to monitor a run by viewing the instrument s progress through the thermal profile of the experiment To monitor the run status 1 Connect to the running instrument 2 Open the Run Status screen In the center of the screen is a representation of the thermal profile for the experiment As an experiment is running the progression of the experiment through the thermal profile protocol is indicated on the display The top of the screen lists the time remaining in the run and the current temperature The bottom of each segment shows how many cycles of the segment have been completed Monitor a run by viewing the raw data plots The Raw Data Plots screen a
134. e first segment in the thermal profile The program marks the segment with a number 1 in the upper left corner as in the example image shown below Agilent AriaMx Real Time PCR Program Setting Up the Thermal Profile 7 2 Click on the segment type that you want to be the second segment in the thermal profile The program marks the segment with a number 2 in the upper left corner 3 Continue clicking on segments to build the thermal profile If you need to redo an assignment click Start Over When finished building the thermal profile click Done The program generates the thermal profile based on your selections and displays it on the Thermal Profile screen 4 Edit the thermal profile as needed Agilent AriaMx Real Time PCR Program 139 7 Setting Up the Thermal Profile Export the thermal profile image The image of the thermal profile can be exported to a Microsoft PowerPoint presentation To export an image of the thermal profile to PowerPoint e From the Thermal Profile screen right click anywhere on the display In the short cut menu click Send Image to PowerPoint PowerPoint opens to a new presentation file with the thermal profile image on the slide 140 Agilent AriaMx Real Time PCR Program Running and Monitoring Experiments Overview of the Instrument Explorer dialog box 142 Add instruments to your network 143 Add a new instrument based on its IP address 143 Add a new instrument based on its
135. e for monitoring from another PC To stop monitoring a run do one of the following e Close the experiment e From the Run Status or Raw Data Plots screen click Stop Monitor In the message box that opens click Yes to confirm that you want to stop monitoring the run Agilent AriaMx Real Time PCR Program 153 Running and Monitoring Experiments Retrieve run data from the instrument 154 If you are monitoring a run from your PC when the run completes the instrument will automatically transfer the run data to the PC program unless you logged into the instrument using the guest account If you are not monitoring the run the data from the run is saved to the instrument and you must retrieve it to your PC in order to analyze the experiment results You can retrieve the data through the network or by copying it to a USB drive Retrieve data through the network If the instrument is connected to the same network as your PC you can retrieve data by connecting to the instrument remotely To retrieve the data from a run by remotely connecting to the instrument from your PC 1 Make sure the experiment file is not open on the instrument 2 From your PC click Instrument gt Instrument Explorer The Instrument Explorer dialog box opens 3 Locate the instrument on which you ran the experiment and in that row click the File Explorer icon pO The File Explorer dialog box opens If this is the first time you have connected to
136. e front of the instrument 2 On the instrument touchscreen open the folder with the post run experiment file 3 Copy the experiment to the USB drive After successful transfer of the file delete the file from the instrument to avoid filling the instrument s hard drive Remove the USB drive from the instrument and insert it into your PC Move the experiment file to a folder of your choice 6 Open the experiment file in the AriaMx program to view the results of the run Agilent AriaMx Real Time PCR Program 155 8 Running and Monitoring Experiments Export instrument data to a CSV file For each post run experiment the program stores the raw data for all dyes in all wells at each data collection point To view this data export it to a CSV file and then open it in Excel Export instrument data by column To export instrument data by column 1 Click Instrument gt Export Instrument Data gt By Columns The Export Instrument Data By Columns dialog box opens 2 Select a folder and file name for the CSV file and click Save The program generates the file and opens it in Microsoft Excel Export instrument data by target To export instrument data by target 1 Click Instrument gt Export Instrument Data gt By Target The Export Instrument Data By Target dialog box opens 2 Select a folder and file name for the CSV file and click Save The program generates the file and opens it in Microsoft Excel Export instrument data by we
137. e is less than the total number of cycles but the amplification plot curve is horizontal Increase in fluorescence in control The reaction has been contaminated reactions without template 326 Agilent AriaMx Real Time PCR Program Reference Help and Troubleshooting amp Support 17 Contact Agilent Technical Support Agilent Technical Support is available worldwide Find the contact information for your country in the table below or on the Genomics website at http www agilent com genomics contactus E mail Telephone Local toll free Americas US and techservices agilent com 800 22 97 0 Canada select options 3 4 3 Brazil chem_vendas agilent com 0800 7281405 Asia and Pacitic Australia agilent_assist agilent com 1800 802 402 Japan email_japan agilent com Q120 4 111 Malaysia ccc smt agilent com 1 800 388 0805 New Zealand agilent_assist agilent com 0508 595 344 Singapore ccc smt agilent com 1800 2 6 2622 South Korea korea inquiry_lsca agilent com 080 004 5090 Europe Austria customercare_Austria agilent com 01 25125 6800 Belgium customercare_Belgium agilent com 02 404 92 22 Denmark customercare_Denmark agilent com 49 0 13 00 30 Finland customercare_Finland agilent com 010 802 220 France customercare_France agilent com 0810 446 446 Germany customercare_Germany agilent com 0800 603 1000 Italy customercare_Italy agilent com 800 0125 5 Netherlands customercare_Netherlands agilent com 020 94 2600 opain customercare spain
138. e map 74 Select wells in the plate map 74 Unselect wells in the plate map 5 View details of a well or wells 75 Export the plate map image 80 Assign plate properties for a Quantitative PCR DNA Binding Dye experiment Assign well types 81 Assign well names 81 Assign dyes targets 82 Select a reference dye 83 Assign replicates 84 Assign quantities to Standard wells 86 Agilent AriaMx Real Time PCR Program 65 81 Assign plate properties for a Quantitative PCR Fluorescence Probe experiment Assign well types 88 Assign well names 88 Assign dyes targets 89 Select a reference dye 90 Assign replicates 91 Assign quantities to Standard wells 93 Assign plate properties for a Comparative Quantitation experiment 95 Assign well types 95 Assign well names 96 Assign sample names and biological replicates 96 Assign dyes targets 98 Select a reference dye 99 Designate the normalizer 99 Assign replicates 100 Assign quantities to Standard wells 102 Assign plate properties for an Allele Discrimination DNA Binding Dye experiment Assign well types 104 Assign well names 105 Assign sample names 105 Assign dyes targets 106 Select a reference dye 107 Assign replicates 108 88 104 Assign plate properties for an Allele Discrimination Fluorescence Probe experiment 111 Assign well types 111 Assign well names 112 Assign sample names 112 Assign dyes targets 113 Select a reference dye 114 Assign Alleles 115 Assign replicates 115 6 Agilent A
139. e of the baseline function is calculated for every cycle and subtracted from the raw fluorescence to produce the baseline corrected fluorescence dR Quantification Cycle Cq The Cq is the cycle at which fluorescence is determined to be statistically significant above background signal contributed by the fluorescently labeled oligonucleotides within the PCR reaction The quantification cycle is inversely proportional to the log of the initial copy number Background Cycle Range The background cycle range specifies the range of cycles of fluorescence data the program uses to calculate the background noise level when using the Background based threshold algorithm to set the threshold fluorescence The region specified is typically in the cycle range before exponential amplification occurs The standard deviation of the raw fluorescence for the specified cycles is calculated and is multiplied by the constant Sigma multiplier for threshold fluorescence Replicates In the AriaMx program the term replicates refers to technical replicates Technical replicates are QPCR reaction tubes containing identical reaction components and set up using a template from the exact same biological sample source While biological replicates measure the variability in the experimental results due to uncontrolled biological variation from sample to sample technical replicates are used to measure the variability in results that is introduced during the process of exp
140. e of the plate map to PowerPoint e From the Plate Setup screen right click anywhere on the plate map In the short cut menu click Send Image to PowerPoint PowerPoint opens to a new presentation file with the plate map image on the slide 80 Agilent AriaMx Real Time PCR Program Setting Up the Plate 6 Assign plate properties for a Quantitative PCR DNA Binding Dye experiment The controls on Plate Setup screen s Properties panel allow you to create a customized plate setup for experiments of the type Quantitative PCR DNA Binding Dye Including Standard Melt To open the Plate Setup screen When you create a new Quantitative PCR experiment you will automatically be directed to the Plate Setup screen To return to the Plate Setup screen at any time before during or after a run click Plate Setup in the Experiment Area panel on the left side of the screen Assign well types Use the Well Types drop down list to assign well types to all the wells used in the experiment See Well types for Quantitative PCR experiments on page 57 for a description of the available well types To assign well types 1 On the Plate Setup screen select all the wells in the plate map that are of the same type For instructions on well selection see Select and view wells in the plate map on page 74 2 Select a well type from the Well Types drop down list in the Properties panel When the Show setting on the Properties panel is set t
141. e results table so that as you scroll through the table horizontally the frozen columns are always visible Right click on the header of the right most column that you want to freeze and click Freeze Column To unfreeze right click again and click Unfreeze Column Display options When you have multiple graphs selected for viewing on the Graphical Displays screen you can manually drag and drop the graphs to new positions on the screen using your cursor the Manual Arrange feature Alternatively you can select for the program to automatically arrange the graphs based on the desired number of graphs per screen the Auto Arrange feature Agilent AriaMx Real Time PCR Program 169 10 Viewing Graphical Displays of the Results 170 To access the options for manually and automatically arranging the graphs click the icon shown below at the bottom of the Graphical Display screen A The following menu opens The options under Manual Arrange and Auto Arrange are described below Manual Arrange Manual Arrange Under Manual Arrange you have two arrangement options Floating arrangement This option allows you to move the graphs to any location on the screen by dragging and dropping them with your cursor Cascade arrangement This option sets the graphs in a cascading arrangement You can move the graphs by dragging and dropping with your Cursor Auto Arrange Under Auto Arrange the options determine the number of grap
142. eased cells or tissues For many gene expression studies your experiments do not require you to determine the absolute amount of a target in a particular sample evidence of a relative increase or decrease in expression compared to a sample of reference is sufficient The sample of reference is referred to as the calibrator For example in a study in which a large number of compounds are screened for the ability to induce the expression of a certain set of genes in HeLa cells the calibrator might be a nucleic acid sample isolated from an untreated HeLa cell culture In a study involving the expression of a cancer marker gene the calibrator might be a nucleic acid sample isolated from the normal non diseased part of the organ whereas the test samples referred to in the program as Unknowns are nucleic acids isolated from the diseased tissue of the same patient The expression level of the target of interest i e the gene you are studying in the calibrator is defined as 1x or 1 0 Expression levels in all unknown samples are reported as a fold difference relative to this calibrator benchmark Normalizing chance variations in target levels The quantity of a target of interest present across a set of independently isolated samples is subject to many variables such as sample to sample differences in total amount of input nucleic acid and differences in the efficiency of RNA extraction or reverse transcription You can include a normalizer t
143. ect Open a new tab and on the Getting Started screen click Multiple Experiment Analysis Click Add Experiment to add the first experiment to the project Click Add Experiment again for each additional experiment that you want to add to the project You can also add experiments after you create the project Enter a name for the project and click Create 2 Set the analysis criteria Navigate to the Analysis Criteria screen Select the wells and targets to include in the analysis and specify the treatment of replicate wells If applicable select the data collection marker to use for analysis only available when toggle button is set to display only one experiment in the project 3 Analyze the data Navigate to the Graphical Displays screen View the results of the analysis and customize analysis settings for individual graphs 4 Export the results To generate a report of the results navigate to the Generate Report screen Configure and create the report according to your selections To export numerical data from the project navigate to the Export Data screen Select the file type and information you want to export Agilent AriaMx Real Time PCR Program Creating and Setting Up an MEA Project 12 Overview of multiple experiment analysis Multiple experiment analysis MEA is a feature in the AriaMx software that allows you to display and analyze the results from two or more post run experiments together in a sing
144. ect Manual if not already selected to turn off the Autoscale functionality 3 In the Min field type the minimum value for the axis 4 In the Max field type the maximum value for the axis 5 Click Close Agilent AriaMx Real Time PCR Program Viewing Graphical Displays of the Results 10 The dialog box closes and the program adjusts the scale of the axis according to your entries To allow the program to automatically set the scale on an axis 1 Open the Graph Properties dialog box to the Axis Options tab 2 Under the heading for the desired axis next to Autoscale select Autoscale if not already selected The Autoscale functionality causes the program to automatically set the range of the axis based on the data points present on the graph 3 Click Close The dialog box closes and the program automatically adjusts the scale of the axis To set the orientation of an axis 1 Open the Graph Properties dialog box to the Axis Options tab 2 Under the heading for the desired axis next to Reverse Orientation select On to plot the values on the selected axis in reverse descending order or select Off to plot the values or ascending order 3 Click Close The dialog box closes and the program adjusts the axis of the graph according to your selection Customize graph grid lines The tools on the Grid Options tab of the Graph Properties dialog box allow you to customize the appearance of the graph s grid lines To select
145. ed in the Last Cycle field specifies which cycle the program uses for the fluorescence values plotted on the graph The last cycle selection only affects the Allele Determine graph It does not affect the amplification plots or the Cq values calculated by the program By default the cycle number in this field is the last cycle of the segment being analyzed but you can specify a different cycle number Agilent AriaMx Real Time PCR Program 209 10 Viewing Graphical Displays of the Results To adjust the last cycle 1 Select the Allele Determination graph on the Graphical Displays screen 2 Inthe right panel click the downward arrowhead above the result table to display the advanced analysis parameter settings 3 Inthe field next to Last Cycle type in the desired cycle number or click the buttons to adjust the cycle number in the field Rename the genotype groups By default the genotypes are given the default names Allele A for the allele A homozygous group Allele B for the allele B homozygous group and Heterozygous for the A B heterozygous group You can assign new names to the genotype groups To assign new names to the genotype groups 1 Select the Allele Determination graph on the Graphical Displays screen 2 Inthe right panel click the downward arrowhead above the result table to display the advanced analysis parameter settings 3 Inthe fields below Rename Genotypes type the desired name for each genotype grou
146. ee Export experiments from the database on page 289 2 Create the project in the same manner as that used for the standard version of the AriaMx software See Create an MEA project on page 240 294 Agilent AriaMx Real Time PCR Program Help for the AriaMx ET Electronic Tracking Software 16 Manage users in the AriaMx ET software If you are running the electronic tracking ET version of the AriaMx software and you logged in using an administrator account you have access to User Management dialog box for managing users and account settings To open the User Management dialog box At the top of the program window click Admin gt User Management Set account properties for all users The General Settings tab of the User Management dialog box has settings that apply to all users on the database General Settings User Settings General Properties Passwords Prompt for password change afterevery 90 days Secure Logout mj Auto lockout after idle for 60 minutes E Signature a Enable E 5ignature Archive Restore g Enable restore and auto rename of experiment with same name as experiment already in Primary database These settings apply to all users Restore Default Settings Agilent AriaMx Real Time PCR Program 295 16 296 Help for the AriaMx ET Electronic Tracking Software Set the password expiration properties You can change how frequently the program prompts users to res
147. een s Properties panel allow you to create a customized plate setup for experiments of the type Allele Discrimination DNA Binding Dye Including High Resolution Melt To open the Plate Setup screen When you create a new Allele Discrimination experiment you will automatically be directed to the Plate Setup screen To return to the Plate Setup screen at any time before during or after a run click Plate Setup in the Experiment Area panel on the left side of the screen Assign well types Use the Well Types drop down list to assign well types to all the wells used in the experiment See Well types for Allele Discrimination DNA Binding Dye experiments on page 63 for a description of the available well types To assign well types 1 On the Plate Setup screen select all the wells in the plate map that are of the same type For instructions on well selection see Select and view wells in the plate map on page 74 2 Select a well type from the Well Types drop down list in the Properties panel When the Show setting on the Properties panel is set to Type the well type appears at the top of the selected wells 3 Repeat steps 1 and 2 for all well types to be included in the experiment 104 Agilent AriaMx Real Time PCR Program Setting Up the Plate 6 Assign well names After you assign wells to a well type you can if desired assign custom well names Show Type Pitas Sample Well Name c u y S
148. efined experiments Contains a complete reaction mixtures with a template sample that is a positive control for mixture of allele A and allele B Calibrator Available in Comparative Quantitation experiments and User Defined experiments as the reference sample to which Unknowns are compared Contains a complete reaction mixture including a characterized target The level of a target of interest in the Calibrator wells is set to 1 0 for comparison to the relative quantities in unknown samples Agilent AriaMx Real Time PCR Program Reference Help and Troubleshooting amp Support 17 Analysis Terms Standard Curve The Standard Curve is a plot of the Cq quantification cycle on the Y axis versus the initial template quantity added to standard wells on the X axis A best fit curve is displayed for each dye with data collected in Standard wells Amplification Plot The Amplification Plots view shows a plot of fluorescence versus cycles for each plateau on which data are gathered Initial Template Quantity Provides interpolated quantities of template added to Unknown wells before thermal cycling The quantities are interpolated from a standard curve based on the calculated Cq values determined for the known quantities of template in the Standard wells Baseline Correction For each well and each path the raw fluorescence data are fit over the specified range of cycles using a linear least mean squares algorithm to produce a baseline The valu
149. ells identified as replicates in the plate setup can be treated either individually each well separate or collectively replicate wells averaged at each cycle during analysis You can choose a treatment for replicate wells from the Analysis Criteria screen or from the Graphical Displays screen To open the Analysis Criteria screen Click Analysis Criteria in the Experiment Area panel on the left side of the screen To specify that replicates be treated individually or collectively e On the Analysis Criteria screen click the Replicates toggle button at the bottom of the screen When the button looks like the image on the left the program treats them individually When the button looks like the image on the right the program treats them collectively es s 162 Agilent AriaMx Real Time PCR Program Setting Analysis Criteria 9 Assign an HRM calibration plate For an experiment that includes a high resolution melt HRM segment in order to view the Melt Curve Difference Plots graph the program requires you to assign an HRM calibration plate HCP to the experiment In order to assign an HCP to an experiment the HCP must e pass the system s quality check e be run on the same instrument as your experiment with the HRM segment e have an identical soak time and plateau time in the HRM segment as the experiment You must retrieve the HCP experiment file from the instrument and save it to your PC using a USB drive For HCP ex
150. en 40 Tools available on the Getting Started screen 41 About the AriaMx file types 42 Quick Start Protocol 43 How to create set up run analyze and generate reports for an experiment 43 Create anew experiment 46 Create an experiment based on experiment type 46 Create an experiment from a template 47 Open an existing experiment 48 Save a copy of an existing experiment 49 Create a template from an existing experiment 50 Convert an experiment to a new experiment type 51 Agg Agilent Technologies 4 Creating Opening an Experiment Overview of the Getting Started screen The tools on the Getting Started screen allow you to create a new experiment from scratch or from a template create a new multiple experiment analysis project or open an existing experiment or project To open the Getting Started screen At the top of the program window click File gt New Alternatively click the icon to open a new tab in the program The new tab opens to the Getting Started screen A Agilent Araki File Instrument Getting Started gt Hew af Experiment Experiment Types Quantitative PCR DHA Binding Dye Including Standard Mett Mey Templates Allele Discrimination DHA Binding Dye including High Resolution Melt Nev Project Mul Ene Experiment Aruaiypls Comparative Quantitation Recentby pensd Experiment Name Experiment I Braat Quantitative PCR Fluorescence Probe Allele Discrimination Fluores
151. en and making changes to the settings click the arrow next to the Save icon to expand the drop down list 2 Click Save The program saves the changes that you made to the settings to the loaded definition To save changes to the data export settings by creating a new definition 1 After loading the saved definition on the Export Data screen and making changes to the settings click the arrow next to the Save icon to expand the drop down list 2 Click Save As The Add New Definition dialog box opens 3 In the Definition Name field type a name for the definition or use the name provided 4 Click Add The dialog box closes and the program saves the data export settings to the new definition Agilent AriaMx Real Time PCR Program 275 14 Generating Multiple Experiment Analysis Reports and Exporting Results Export MEA data results to an Excel or text file 276 Agilent AriaMx Real Time PCR Program 15 How To Examples for Multiple Experiment Analysis Projects Example1 2 8 How to find the initial template quantity of a target in an unknown sample using a standard curve from a separate experiment 2 8 Example 2 280 How to normalize target quantities in Unknown and Calibrator wells using a normalizer target from a separate experiment 280 fhe Agilent Technologies 15 How To Examples for Multiple Experiment Analysis Projects Example 1 How to find the initial template quantity of a target in an
152. ene called a normalizer that is known to be unaffected by the experimental conditions such as a housekeeping gene For more information see The Comparative Quantitation Experiment Type on page 58 Agilent AriaMx Real Time PCR Program Selecting an Experiment Type 5 Allele Discrimination Fluorescence Probes The Allele Discrimination experiment type is used to discriminate between two alleles in a DNA sample For more information see The Allele Discrimination Fluorescence Probe Experiment Type on page 64 The program offers two sub types for the Allele Discrimination experiment type In the sub type Fluorescence Probe you use two fluorogenic probes labeled with different dyes to discriminate between two alleles in a DNA sample For example if amplification in an unknown DNA sample is detected for the dye identifying the wild type allele but not for the dye identifying a mutant allele the sample can be designated as wild type homozygous Allele Discrimination DNA Binding Dye with High Resolution Melt The Allele Discrimination experiment type is used to discriminate between two alleles in a DNA sample For more information see The Allele Discrimination DNA Binding Dye Experiment Type on page 62 The program offers two sub types for the Allele Discrimination experiment type In the sub type DNA Binding Dye Including High Resolution Melt you use SYBR Green or EvaGreen dye to detect both alleles and incl
153. enerate Report screen and making changes to the report configuration click the arrow next to the Save icon F to expand the drop down list 2 Click Save The program saves the changes that you made to the report configuration to the loaded definition To save changes to a report configuration by creating a new definition 1 After loading the saved definition on the Generate Report screen and making changes to the report configuration click the arrow next to the Save icon Fi to expand the drop down list 2 Click Save As The Add New Definition dialog box opens 3 In the Definition Name field type a name for the definition or use the name provided 4 Click Add The dialog box closes and the program saves the report configuration to the new definition Agilent AriaMx Real Time PCR Program 227 11 Generating Reports and Exporting Results Export data results to an Excel text or RDML file 228 The Export Data screen has tools for exporting numerical data from the experiment to an Excel text or RDML file Data files can include data on the setup of the experiment as well as data from the results of the experiment To open the Export Data screen Click Export Data in the Experiment Area panel on the left side of the screen Configure the file and export data Before creating the file you can configure the file by selecting which data items to include If desired you can save the configuration as a data
154. er of plateaus in the thermal profile according to your selection A 3 step amplification segment has 3 plateaus one for denaturation one for annealing and one for extension A 2 step amplification segment has only 2 plateaus one for denaturation and a combined annealing extension plateau Agilent AriaMx Real Time PCR Program Setting Up the Thermal Profile 7 To change the number of cycles for an Amplification segment 1 On the display click directly on the number of cycles shown at the bottom of the segment The cycle number becomes an editable field 2 Type the desired number of cycles into the field or click the buttons until you reach the desired number 3 Press Enter or click anywhere outside of the field Edit data collection markers To add a data collection marker 1 On the display hover the cursor over the plateau or ramp where you want to add a new data collection marker A grayed out image of a data collection marker appears on the display 2 Click the image of the data collection marker The program adds the data collection marker to the thermal profile Only melt segments can have a data collection marker assigned to a ramp To remove a data collection marker e On the display click directly on the data collection marker that you want to remove The program removes the data collection marker from the thermal profile Edit ramp properties melt segments only If you are collecting data duri
155. eriment Types 54 Quantitative PCR 54 Comparative Quantitation 54 Allele Discrimination Fluorescence Probes 55 Allele Discrimination DNA Binding Dye with High Resolution Melt 55 User Defined 55 The Quantitative PCR Experiment Type 56 Multiplexing quantitative PCR experiments 56 Well types for Quantitative PCR experiments 5 The Comparative Quantitation Experiment Type 58 Normalizing chance variations in target levels 58 Determining amplification efficiencies for the targets of interest and normalizer targets 59 Including biological replicates in comparative quantitation 60 Well types for Comparative Quantitation experiments 61 The Allele Discrimination DNA Binding Dye Experiment Type 62 About HRM calibration plates 62 Well types for Allele Discrimination DNA Binding Dye experiments 63 The Allele Discrimination Fluorescence Probe Experiment Type 64 Well types for Allele Discrimination Fluorescence Probe experiments 65 The User Defined Experiment Type 66 Agg Agilent Technologies 5 Selecting an Experiment Type Overview of Experiment Types 54 The AriaMx program offers a variety of experiment types Each experiment type was designed for a specific application and has its own unique options for experimental setup and analysis that are specialized for that application The AriaMx experiment types are summarized below Quantitative PCR The Quantitative PCR experiment type is the best choice when you need to de
156. erimental setup Designating replicate wells allows the program to Agilent AriaMx Real Time PCR Program 321 17 322 Reference Help and Troubleshooting amp Support average results from those wells when the Treat Collectively setting is used Collective Replicate Treatment Collective replicate treatment causes the program to analyze all wells with the same replicate symbol as a group effectively treating the measurements as all coming from the same well Biological Replicates Biological replicates are template samples that were isolated independently but from biologically identical sources that is sources that are genetically identical are of the same cell type and were treated identically during experimentation For example two samples of cDNA that were isolated from the same tissue source in two different mice that were exposed to identical conditions and have the same genotype would be biological replicates Biological replicates help you determine the level of variability in gene expression for your specific experiment that is due to uncontrolled biological variation from sample to sample When setting up the plate for a Comparative Quantitation experiment you may designate two or more samples as biological replicates while assigning the sample names Samples that are biological replicates are assigned the same sample name but have different biological replicate ID numbers R squared The R value is an indication of the fit
157. ermination graph on page 206 By default data from all of the wells that you selected on the Analysis Criteria screen are displayed in the graphs You can limit the graphs to only display data from particular wells or replicate sets by selecting specific rows in the result table press Ctrl to select multiple rows The result table is described below Agilent AriaMx Real Time PCR Program Viewing Graphical Displays of the Results 10 Result table The result table on the right side of the Graphical Displays screen shows results for each well if replicates are treated individually or replicate set if replicates are treated collectively that you selected on the Analysis Criteria screen see Select the wells and well types to include in analysis on page 159 Row selection You can select individual rows within the results table to limit the graphs to displaying data only from particular wells replicate sets Click directly on a row to select it Press Ctrl to select multiple rows By default all rows are initially selected Click the Select All icon 2 at the top of the result table to reselect all rows Data columns You can configure which columns of data are included in the table Click the Column Options icon ef at the top of the result table to open the Column Options dialog box In the dialog box mark the columns that you want to include in the result table You can freeze one or more columns on the left side of th
158. erview of the Analysis Criteria screen 158 Select the wells and well types to include in analysis Select wells for analysis using the plate map 159 Select wells for analysis based on well type 159 Select the targets to include in analysis 160 Select which data collection points to analyze 161 Choose a treatment for replicate wells 162 Assign an HRM calibration plate 163 Assign an HCP for new experiments 163 Assign an HCP using the HCP icon 164 10 Viewing Graphical Displays of the Results 167 Overview of the Graphical Displays screen 168 Graphs 168 Result table 169 Display options 169 Zooming 171 View the Amplification Plots 173 View data for a single data point 173 151 159 Agilent AriaMx Real Time PCR Program Select a fluorescence datatype 1 4 Specify the use of smoothing 1 4 Adjust the graph properties 1 5 Adjust the baseline correction settings 1 5 Select the scale linear or log of the Y axis 177 Manually adjust threshold fluorescence values 177 Adjust threshold fluorescence values by altering the algorithm settings 180 Lock or unlock the threshold fluorescence values 181 View the Melt Curve Raw Derivative Curve 183 About raw derivative curves 183 View data for a single data point 184 Select a fluorescence datatype 184 Adjust the graph properties 185 Specify the use of smoothing 185 Normalize the fluorescence values 185 Adjust the range of the X axis 186 Adjust product melting temperature settings 187
159. es Each bar on the graph is a target within a well or replicate set The expression level of the target of interest in the Calibrator wells is defined 1 0 In the Unknown wells the expression levels of the target of interest are reported on the Y axis as a fold difference relative to the calibrator benchmark How you set up the wells on the Plate Setup screen impacts how the program compares samples in the Relative Quantity chart e If you designate a separate calibrator sample for each unique target the program only compares Unknown wells with that same target to those Calibrator wells This allows you to run multiple targets on the same plate and analyze them independently e If you set up multiple Calibrator wells replicate sets with the same target name the program will average the Cq values of those Calibrator wells and calculate the relative quantities of that target in the Unknown wells relative to the average Agilent AriaMx Real Time PCR Program 201 10 Viewing Graphical Displays of the Results 202 Select a fluorescence data type For the Y axis of the Relative Quantity chart you can use baseline corrected fluorescence values with or without normalization to a reference dye To select the type of fluorescence data displayed in the Relative Quantity chart 1 Select the Relative Quantities chart on the Graphical Displays screen 2 Inthe right panel select one of the options next to Fluorescence Term The possible op
160. et name to the selected wells If you do not assign a target name the program uses the dye name as the target name 5 Repeat steps 1 4 for all wells included in use in the experiment Agilent AriaMx Real Time PCR Program 121 6 Setting Up the Plate 6 Optional Select a new color to associate with a target a Click the colored dot to right of the Target Name field b In the selection box that opens click the desired color or click Advanced for more color options Select a reference dye You can include a reference dye e g ROX dye to normalize the fluorescence signal of the reporter dye To assign a reference dye e On the Plate Setup screen select the reference dye from the Reference Dye drop down list in the Properties panel The program assigns the target name REF to all wells in use in the experiment and displays an R in the wells of the plate map to indicate that the well contains a reference dye Designate the normalizer In order to normalize the quantity level of your target of interest to a normalizer target you need to amplify the normalizer in both the Unknown wells and the Calibrator wells You can amplify the normalizer in the same well as the target of interest if the two targets are detected with spectrally distinct dyes or you can amplify them in separate wells that contain the same template sample To assign a normalizer target 1 On the Plate Setup screen select the wells that will be u
161. et their passwords You can also eliminate password expirations so that the program no longer requires users to reset their passwords after a set period of time To change the password expiration time 1 Open the User Management dialog box to the General Settings tab 2 Under Passwords make sure the check box is marked and in the field type the number of days that you want the expiration time to be The default is 90 days which means that the program automatically prompts users to reset their password every 90 days 3 Click Close to close the dialog box and apply your changes To eliminate password expirations 1 Open the User Management dialog box to the General Settings tab 2 Under Passwords clear the check box next to Prompt for password change every x days 3 Click Close to close the dialog box and apply your changes Users will no longer be prompted to change their passwords after a set period of time Set the auto lockout properties You can change the length of idle time required before the program automatically locks and requires users to re enter their password You can also disable the automatic lockout feature completely To change the length of idle time required before automatic lockout 1 Open the User Management dialog box to the General Settings tab 2 Under Secure Logout make sure the check box is marked and in the field type the number of minutes for the desired idle time The default is 60 minutes w
162. ets of wells on the plate map Undo Redo e Click the Undo icon in the lower right corner of the screen to undo your most recent action You can click the icon multiple times to undo multiple consecutive actions Click the Redo icon in the lower right corner of the screen to redo the most recent action that you reversed using the Undo tool You can click the icon multiple times to redo multiple consecutive actions You can also access the Undo and Redo commands by right clicking anywhere on the plate map Clear Selected Well or Clear Plate To clear all the properties assigned to a well or set of wells select the well s on the plate map right click and then click Clear Selected Well Alternatively select the well s on the plate map and press Delete to clear the properties To clear all properties assigned to all wells in the plate right click anywhere on the plate map and click Clear Plate Agilent AriaMx Real Time PCR Program Setting Up the Plate 6 Import a plate setup The AriaMx program allows you to set up the plate on the Plate Setup screen by importing the plate setup of any existing experiment of the same experiment type After you import the plate setup you can edit the well properties as desired To open the Plate Setup screen With an experiment or project file open click Plate Setup in the Experiment Area panel on the left side of the screen To import a plate setup 1 In the Properties panel of t
163. ew cycle range and adds an asterisk to the Well column to indicate that the cycle range for that plot differs from the adaptive values To set the baseline cycle range back to adaptive values 1 Open the Baseline Correction dialog box 2 Select the plots a plot is one target in one well that you want to reset to the adaptive values by clicking the appropriate line in the table Press Ctrl while clicking to select more than one plot Click Select All to select all plots in the table 3 Click Reset The program updates the cycle ranges listed in the table back to the adaptive values Select the scale linear or log of the Y axis By default the fluorescence values on the Y axis of the amplification plots are in linear scale You can quickly toggle between linear scale and log scale from the Graphical Displays screen To select the scale of the Y axis 1 Select the Amplification Plots graph on the Graphical Displays screen 2 In the right panel click the downward arrowhead above the result table to display the advanced analysis parameter settings 3 Next to Graph Type select Linear to plot the fluorescence values on a linear scale or select Log to plot the fluorescence values on a log scale The program adjust the amplification plots according to your selection Manually adjust threshold fluorescence values The threshold fluorescence value for a target determines the target s Cq value The Cq is the cycle number at which t
164. experiment Marking a snapshot will export only that snapshot The date stamp for the snapshots are noted in the Date column 3 Click Browse The Browse For Folder dialog box opens 4 Browse to the folder where you want to save the exported experiments Select the folder and click Open The Browse For Folder dialog box closes and the file path for the selected folder is listed in the Export To Folder field on the Export Experiments dialog box 5 Click Export The dialog box closes The program creates a copy of the experiment and saves it to the specified folder The file name of the exported experiment includes a date and time stamp 290 Agilent AriaMx Real Time PCR Program Help for the AriaMx ET Electronic Tracking Software 16 Lock or log out of the AriaMx ET software When the Auto lockout feature is enabled the program automatically locks and requires users to re enter their password if the program is idle for longer than a set period of time 60 minutes is the default You can also lock the program or log out completely For example if you need to step away from your computer or need to secure it for any reason you may want to lock the program or log out Lock the program Locking the program makes that session of the program inaccessible without logging out the currently logged in user To unlock the program users must re enter the user password Only the logged in user or an administrator can unlock a locked sessio
165. export definition which you can later load with a future experiment see Load a saved data export definition Select the items to include in the file To include and exclude item from the file e In the Export Configuration Panel of the Export Data screen under Items mark the check boxes for the items that you want to include in the file Clear the check boxes for any items that you do not want to include The preview of the file in the center of the screen includes only the marked items Above each page is the name of the item To customize the elements of the Plate Setup Thermal Profile Tabular Results or Experiment Notes 1 Inthe Export Configuration Panel of the Export Data screen under Items make sure that the check box for the item that you want to customize is marked 2 Click the icon next to the item The Column Options dialog box opens 3 Mark the check boxes for the elements that you want to include in the exported file Clear the check boxes for elements that you do not want to include 4 Click OK in the Column Options dialog box Agilent AriaMx Real Time PCR Program Generating Reports and Exporting Results 11 The dialog box closes and the preview on the Export Data screen includes your changes Export to Excel When you export data results to Excel the program automatically launches Microsoft Excel and creates a workbook with each data item displayed on a separate tab within the workbook You can the
166. ext to Show select Name The Well Name field becomes available for typing 2 Select all the wells in the plate map that you want to assign to the same well name For instructions on well selection see Select and view wells in the plate map on page 74 You must assign a well to a well type before you can assign it a well name 3 In the Well Name field type the well name for the selected wells Press Enter The Well Name appears at the top of the selected wells 4 Repeat steps 2 3 for all well names that you want to assign Assign sample names and biological replicates Once well types have been assigned you can specify the sample in each well by assigning sample names Each unique template sample included in the experiment can be assigned its own sample name If your experiment ts designed for comparative quantitation and the normalizer target and the target of interest are being amplified in different wells be sure to assign the same sample name to these wells so the data are normalized properly during analysis If your experiment is designed for allele determination and the two alleles are being amplified in separate wells the sample name is used to associate the wells amplifying Allele A with the wells amplifying Allele B from the same template For samples that are biological replicates assign the same sample name to the wells but give the wells different biological replicate ID numbers to keep them differenti
167. f you are working from the touchscreen version of the program a Load your reaction plate into the instrument s thermal block b On the Thermal Profile screen press Run Experiment The instrument starts running the experiment You can monitor the progress of the run from your PC See Monitor a run on page 149 for instructions Cancel a run To stop an in progress run 1 Make sure you are monitoring the run See Monitor a run on page 149 2 On the Run Status or Raw Data Plots screen click Stop Run A message box opens asking you to confirm that you want to stop the run 3 Inthe message box if you do not want to save the partial data collected during the run mark the check box labeled Discard Instrument Data If you do want to save the partial data leave the check box unmarked 4 Click Abort Experiment in the message box to stop the run If you chose to save the partial data you can view it on the Graphical Displays screen Agilent AriaMx Real Time PCR Program 147 148 Running and Monitoring Experiments Pause a run To pause an in progress run 1 Make sure you are monitoring the run See Monitor a run on page 149 2 On the Run Status or Raw Data Plots screen click Pause A message box opens asking you to confirm that you want to pause the run 3 Inthe message box click Pause Run The program pauses the run When you are ready to resume running the experiment click Resume Agilent Aria
168. fidence interval The program displays the hashed lines on the Standard Curve graph indicating the confidence intervals If you had selected to treat replicates collectively the program will prompt you to treat them individually in order to display the intervals To adjust the confidence level 1 Display the confidence intervals on the Standard Curve graph see instructions above 2 Next to Level type the desired confidence level into the field or click on the buttons to adjust the value in the field The program updates the positions of the confidence interval lines on the graph to reflect the new confidence level 200 Agilent AriaMx Real Time PCR Program Viewing Graphical Displays of the Results 10 View the Relative Quantity For Comparative Quantitation experiments and User Defined experiments that include a set of Calibrator wells the Graphical Displays screen includes a Relative Quantity chart This chart is a bar graph that shows the amount of target present in the experimental samples the Unknown wells relative to the associated reference sample the Calibrator wells after the program has normalized the quantities using data from the normalizer target To view the Relative Quantity Click Graphical Displays in the Experiment Area panel on the left side of the screen If the Relative Quantity chart is not already displayed click the Relative Quantity icon at the bottom of the screen About relative quantiti
169. g able to log in to the AriaMx ET program You can also re enable a disabled user account To disable a user account 1 Open the User Management dialog box to the User Settings tab 2 In the table locate the account that you want to disable and clear the check box in the Enable column 3 Click Close to close the dialog box and apply your changes To enable a user account 1 Open the User Management dialog box to the User Settings tab 2 In the table locate the account that you want to enable and mark the check box in the Enable column 3 Click Close to close the dialog box and apply your changes Change a user s password As an administrator you can change the password for any user account in the database To change a password 1 Open the User Management dialog box to the User Settings tab 2 In the table click on the row for the desired user account to select it Agilent AriaMx Real Time PCR Program Help for the AriaMx ET Electronic Tracking Software 16 The account information for the selected user is displayed in the bottom panel of the dialog box 3 Click Change Password The Password and Retype Password fields become editable 4 Inthe Password field type a new password for the account Passwords must 6 15 alphanumeric characters in length and contain a number 5 Inthe Retype Password field type the password again 6 Click Save to save the new password Print or export the user account table You can pr
170. g alters the shapes of the curves by decreasing the effects of signal noise The smoothing option is turned on by default To turn smoothing on or off 1 Select the Melt Curve Raw Derivative Curve graph on the Graphical Displays screen 2 Inthe right panel click the downward arrowhead above the result table to display the advanced analysis parameter settings 3 Next to Savitzky Golay select On to turn on smoothing or select Off to turn off smoothing 4 If you selected On next to Number of Points select the number of data points on each melt curve that you want the algorithm to use in the smoothing calculations The options are 5 7 9 and 11 A higher number of points leads to a smoother i e more manipulated curve Normalize the fluorescence values To facilitate comparison among the curves on the graph you can select to normalize the fluorescence values on the Y axis Normalization rescales the Y axis value of each data point in each curve such that the minimum Agilent AriaMx Real Time PCR Program 185 10 Viewing Graphical Displays of the Results 186 fluorescence value for each plot is set to 0 and the maximum fluorescence value is set to 1 The normalization option is turned off by default To turn normalization on or off 1 Select the Melt Curve Raw Derivative Curve graph on the Graphical Displays screen 2 Inthe right panel click the downward arrowhead above the result table to display the advanced analy
171. ght of the Target Name field b In the selection box that opens click the desired color or click Advanced for more color options Agilent AriaMx Real Time PCR Program Setting Up the Plate 6 Select a reference dye You can include a reference dye e g ROX dye to normalize the fluorescence signal of the reporter dye To assign a reference dye e On the Plate Setup screen select the reference dye from the Reference Dye drop down list in the Properties panel The program assigns the target name REF to all wells in use in the experiment and displays an R in the wells of the plate map to indicate that the well contains a reference dye Designate the normalizer In order to normalize the quantity level of your target of interest to a normalizer target you need to amplify the normalizer in both the Unknown wells and the Calibrator wells You can amplify the normalizer in the same well as the target of interest if the two targets are detected with spectrally distinct dyes or you can amplify them in separate wells that contain the same template sample When you designate multiple normalizers in the same well the program first calculates the normalized target of interest levels for each normalizer separately and then calculates the geometric mean of all normalized values The program then uses the geometric mean for the Relative Quantity calculations To assign a normalizer target 1 On the Plate Setup screen select
172. gram 1 Select the Standard Curve graph on the Graphical Displays screen 2 Inthe right panel click the downward arrowhead above the result table to display the advanced analysis parameter settings 3 Under Threshold Fluorescence click the Recalculate icon next to the desired target The program resets the threshold fluorescence value in the field back to the default value and adjusts the standard curves accordingly Agilent AriaMx Real Time PCR Program 199 10 Viewing Graphical Displays of the Results Display and adjust confidence intervals You can select to display the confidence intervals for each plot on the Standard Curve graph as hashed lines These lines show the range of initial quantity values at a particular Cq that cannot be statistically distinguished from the fit line with more certainty than the specified confidence level The width of the confidence interval is an indicator of the quality of the fit of the data to the standard curve The default confidence level is 99 When you select to display the confidence intervals you can adjust the confidence level To display confidence intervals the program must treat replicates individually To display confidence intervals 1 Select the Standard Curve graph on the Graphical Displays screen 2 Inthe right panel click the downward arrowhead above the result table to display the advanced analysis parameter settings 3 Under Threshold Fluorescence mark Show con
173. gt Preferences Fij Preferences Configure Expenment Project Filename New Experiments Project File Naming Custom fg Experiment 1 cancel To configure the default file name for experiments and projects 1 In the Preferences dialog box next to New Experiment Project File Naming click one of the following options e Default Select this option to use the program s default file naming system For experiments the default file name is Experiment followed by the next available number e g Experiment 3 For projects the default file name is Project followed by the next available number e g Project 3 26 Agilent AriaMx Real Time PCR Program Specifying Instrument and Program Settings 2 e Custom Select this option to configure your own file naming system by selecting which pieces of information to include in the default file name Using the check boxes you can select to include the experiment type the creation date for the experiment or project in format MM DD YYYY DD MM YYYY or YYYY MM DD and the time that the experiment or project was created Experiment Type K Date DD MM YYYY YYYY MM DD C Time 2 Click OK to close the dialog box and save your changes Agilent AriaMx Real Time PCR Program 2 2 Specifying Instrument and Program Settings Set program preferences 28 Agilent AriaMx Real Time PCR Program 3 Performing Hardware and Software Tests and HRM Calibr
174. h dyes it designates the unknown sample as heterozygous for the two alleles With properly designed probes this assay is sensitive enough to detect a single base difference single nucleotide polymorphism or SNP between two alleles The program uses the quantification cycle Cq value for each target in each sample to determine the genotypes of the samples A Cq value of 24 to 32 is expected for a sample that contains the specific allele recognized by the probe A Cq value equal to the final cycle of the PCR reaction typically 40 or equal to the Cq of the negative control samples indicates the absence of a specific allele The program displays the results in the Allele Determination graph which is a scatter plot that shows the Cq for the dye specific to one allele plotted against the Cq for the dye specific to the other allele The program groups plotted points according to their positions on the graph providing a convenient visualization of samples which share the same genotype allelic composition In the analysis of real time Allele Discrimination experiments that use fluorescence probes e g TaqMan probes you would typically monitor and report fluorescence at the end of the annealing extension step At that point the polymerase has already extended across the template and hydrolyzed any probe that had annealed Agilent AriaMx Real Time PCR Program Selecting an Experiment Type 5 Well types for Allele Discrimination Fluorescenc
175. he Plate Setup screen click the Import Plate Setup icon ann ae E The Open dialog box opens 2 Browse to the folder of the existing experiment with the plate setup that you want to import Select the file and click Open The dialog box closes and the program imports the plate setup into the currently open experiment Agilent AriaMx Real Time PCR Program 73 6 Setting Up the Plate Select and view wells in the plate map When setting up your plate on the Plate Setup screen you may need to select a sub set of wells in the 96 well plate map in order to assign properties to specific wells You can select and unselect wells by clicking directly on the plate map You may also need to view certain wells of the plate map in more detail which can also be done from the Plate Setup screen To open the Plate Setup screen With an experiment or project file open click Plate Setup in the Experiment Area panel on the left side of the screen Select wells in the plate map When a new experiment is first opened all wells on the plate are already selected Selected wells appear highlighted on the plate map see wells Al through A6 in the image below while unselected wells appear grayed out see wells A7 through A12 in the image below M 1 2 3 4 5 6 T g To select all wells when some wells are not already selected 1 Click the small square in the upper left hand corner of the plate If all wells on the plate are already selected clic
176. he amplification data by target the program generates a separate graph for each target If multiple experiments in the project amplified the same target the program displays the amplification plots from these different experiments on the same graph Make sure each unique target has a unique name See Differentiate between targets across experiments on page 244 This view provides a good way to compare how the same target performed in different experiments Note that comparing Agilent AriaMx Real Time PCR Program Analyzing Multiple Experiment Analysis Project Results 13 amplification plots by target rather than by experiment does not impact the Cq values that the program calculates for each plot To compare by experiment 1 Select the Amplification Plots graph on the Graphical Displays screen 2 Inthe right panel next to Compare select Target Consolidate the amplification plots When you select to consolidate the amplification data the program displays all of the amplification plots from all experiments on a single graph The program calculates the Cq values in the same manner as it does when you compare the data by experiment or by target To consolidate the amplification plots 1 Select the Amplification Plots graph on the Graphical Displays screen 2 Inthe right panel next to Compare select Consolidated Agilent AriaMx Real Time PCR Program 257 13 Analyzing Multiple Experiment Analysis Project Results Compare r
177. he fluorescence level passes the threshold You can manually adjust the threshold fluorescence values while viewing the amplification plots Agilent AriaMx Real Time PCR Program 177 10 Viewing Graphical Displays of the Results To adjust the threshold fluorescence value for a target by typing the desired value 1 2 Select the Amplification Plots graph on the Graphical Displays screen In the right panel click the downward arrowhead above the result table to display the advanced analysis parameter settings Under Threshold Fluorescence type a new value into the field next to the desired target or click on the buttons to adjust the value If the lock icon is in the locked position for the target click on it to unlock before the adjusting threshold See Lock or unlock the threshold fluorescence values on page 181 for more information The position of the corresponding horizontal line on the amplification plots adjusts to the new threshold fluorescence value To adjust the threshold fluorescence value for a target by dragging the threshold line on the Amplification Plots graph 1 178 Select the Amplification Plots graph on the Graphical Displays screen The threshold fluorescence level for each target is displayed on the graph as a horizontal line with a triangle marker along the Y axis In the right panel click the downward arrowhead above the result table to display the advanced analysis parameter settings
178. he reference dye fluoresces at a constant level during the reaction Reporter Dye Fluorescent dye that increases in fluorescence signal as the amount of PCR product increases Normalizer Available in Comparative Quantitation experiments and User Defined experiments the normalizer is a target that is known to be unaffected by the experimental treatment under investigation and thus is found in equal quantity across all template samples Data from the normalizer target is used to normalize the fluorescence signal of the targets of interest FRET Chemistry Fluorescence Resonance Energy Transfer In FRET chemistry the excitation of a donor fluorescent dye is transferred to a receptor dye leading to the fluorescence of the acceptor dye instead of the donor dye The transfer is possible only if the two dyes are in close proximity TaqMan Probes These are linear fluorescently labeled hydrolysis probes that can be used to monitor PCR product formation either during or after the amplification process As the DNA polymerase extends the upstream primer and encounters the downstream probe the exonuclease activity of the polymerase cleaves the probe In this event the reporter fluorophore is released into the reaction solution and is able to fluoresce Quencher A quencher is a moiety that absorbs the energy of the reporter dye in its excited state The quencher can emit its own fluorescence signal TAMRA or emit no fluorescence signal DABCYL
179. hecked items to a genotype Homozygous A Homozygous B or Heterozygous or to remove genotype calls from the checked items Clear Call command This command is used for manual genotype calling in the Melt Curve Difference Plots graphs If you added a check mark to specific wells or replicate sets using the Check Item command or the Check column in the Results Table use this command to remove genotype calls from the checked items This command is used for manual genotype calling in the Melt Curve Difference Plots graphs It opens the HRM Manual Call Settings dialog box which allows you to assign a color and name to each genotype for use in the difference plots This set of four options is only available for Allele Determination graphs Use the options to specify which genotype groups on the graph are visually grouped together with a rectangle See Display genotype groups on the graph on page 208 for more information These two commands are part of the Axis Options sub menu Click directly on one of these commands to toggle between a linear scale and a log scale for the X and Y axes of the graph When a command has a check mark next to it that indicates that the axis is set to a log scale The absence of a check mark indicates that the axis is set to a linear scale These two commands are part of the Axis Options sub menu Click directly on one of these commands to toggle the orientation of the X and Y axes of the graph When a
180. hich means that the program automatically locks after 60 minutes of inactivity 3 Click Close to close the dialog box and apply your changes Agilent AriaMx Real Time PCR Program Help for the AriaMx ET Electronic Tracking Software 16 To disable automatic logout 1 Open the User Management dialog box to the General Settings tab 2 Under Secure Logout clear the check box next to Auto lockout after idle for x minute 3 Click Close to close the dialog box and apply your changes The program no longer locks after a period of inactivity Disable or enable the e signature requirement By default the program requires users to re enter their password before allowing them to print or export an experiment report This step serves as an electronic signature e signature to verify that the logged in user approves of the report The User Management dialog box has tools for disabling and enabling the e signature requirement To disable e signature 1 Open the User Management dialog box to the General Settings tab 2 Under E Signature clear the check box 3 Click Close to close the dialog box and apply your changes The program no longer requires an e signature to print or export reports To enable e signature 1 Open the User Management dialog box to the General Settings tab 2 Under E Signature mark the check box 3 Click Close to close the dialog box and apply your changes The program now requires an e signature to print or
181. hs displayed on the screen 1 2 3 or 4 The image in each icon shows the arrangement of the graphs associated with that option When you select to display more than one graph at a time you can reorder the positions of the graphs by dragging and dropping one graph on top of another with your cursor Agilent AriaMx Real Time PCR Program Viewing Graphical Displays of the Results 10 Zooming Within a graph you can zoom in on a particular region of interest Drag your cursor across the region of interest as shown below Amplification Plots a x 3500 3000 j b B B S S 3 Fluorescence AR 500 The program then zooms in on the selected region Amplification Plots a x S 8 Fluorescence AR z 20 22 24 26 28 30 32 34 36 Cycles Agilent AriaMx Real Time PCR Program 171 10 Viewing Graphical Displays of the Results To reset the zoom level right click anywhere on the graph and click Reset Zoom For more information on the display options available for the graphs on the Graphical Displays screen see Customize graph properties on page 211 172 Agilent AriaMx Real Time PCR Program Viewing Graphical Displays of the Results 10 View the Amplification Plots The Amplification Plots graph on the Graphical Displays screen shows a plot of fluorescence Y axis versus cycles X axis for each target in each well or in each set of replicate wells at a single data collection point The panel on the right si
182. iaMx ET database 313 View transaction logs in the AriaMx ET software 315 View transaction logs for primary database 315 View transaction logs for an archive database 316 17 Reference Help and Troubleshooting amp Support 317 QPCR Glossary 318 Experiment Type and QPCR Detection Chemistry Terms 318 Well Types 320 Analysis Terms 321 Trademarks 324 Troubleshooting and Support 325 Troubleshooting Guide 325 Contact Agilent Technical Support 327 14 Agilent AriaMx Real Time PCR Program Getting Started with the AriaMx Program The AriaMx program 16 Getting Started with the AriaMx Software 17 Introduction to the AriaMx software 17 Overview of the user interface 19 Home screen 19 Menu toolbar 19 Tabs 19 Left and right panels 19 Home Notifications Help icons 20 Getting Started screen 20 Experiment Notes Project Notes 20 Help access for the AriaMx software 21 Help system 21 Video tutorials 22 Sample experiments 22 Agg Agilent Technologies 1 Getting Started with the AriaMx Program The AriaMx program 16 The AriaMx Real Tim PCR software features a variety of experiment type options with customized plate and thermal profile setup and experiment analysis screens that streamline the process of collecting and analyzing data for specific applications The software capabilities allow you to Quickly set up an experiment using a template or the Import Plate Setup function View raw fluorescence data without mathematical cor
183. ienced in QPCR your individual needs are met with the comprehensive and powerful tools AriaMx offers backed by Agilent s industry leading support The AriaMx Real Time PCR System is a fully integrated quantitative PCR amplification detection and data analysis system The system design combines a state of the art thermal cycler an advanced optical system with an LED excitation source and complete data analysis software The instrument is provided with five optics modules and the scanning optics design delivers optimal separation between both dyes and samples The AriaMx provides a closed tube PCR detection format that can be wed with a variety of fluorescence detection chemistries e g SYBR Green and EvaGreen dyes as well as fluorogenic probe systems including TagMan Probes and molecular beacons C Show Home on StartUp License Agilent Technologies Reterved 2014 Contact Support eS a OERE eS Se ee Agilent AriaMx Real Time PCR Program 17 1 18 Getting Started with the AriaMx Program Introduction To view the Introduction click Introduction near the top right corner of the Home screen The text on this screen provides an overview of the AriaMx Real Time PCR System Features To view the Features click Features near the top right corner of the Home screen The text on this screen lists the features of the AriaMx software Agilent AriaMx Real Time PCR Program Getting Started with the AriaMx Program 1
184. ification efficiencies of the normalizer target and the target of interest before you use a particular normalizer in a Comparative Quantitation experiment See the following section Determining the Amplification Efficiencies for the Target of Interest and Normalizer Targets for more information Determining amplification efficiencies for the targets of interest and normalizer targets In developing a Comparative Quantitation experiment it is important that the amplification efficiencies of the target of interest and the normalizer target are reproducible and ideally very similar To measure the amplification efficiency for each target generate a standard curve ina Quantitative PCR experiment When running a standard curve for the sole purpose of determining the amplification efficiency for a particular target it is not necessary to know the exact quantity of your targets Instead you can use serially diluted template as the standard samples and assign the standard quantities in Plate Setup using the relative unit designation The program then analyzes the standard curve data and calculates the amplification efficiency from the slope of the curve See View the R squared values slopes and amplification efficiencies on page 198 for more information on deriving amplification efficiencies from standard curves If differences in amplification efficiency exist In an idealized Comparative Quantitation experiment the amplific
185. iguration Panel of the Generate Report screen under Items make sure that the Cover Page check box is marked Click the icon next to the Cover Page item The Cover Page Options dialog box opens In the Report Title and Report Description fields edit the content as desired The title and description are printed on the cover page Under Other Items mark the check boxes for the pieces of information that you want to include on the cover page Clear the check box for any pieces of information that you do not want to include In the fields labeled Left Center and Right type the text that you want to appear at the bottom of the cover page on the left side center and right side Click OK in the Cover Page Options dialog box The dialog box closes and the Cover Page displayed in the report preview includes your changes To customize the information included in the Tabular Results 1 In the Report Configuration Panel of the Generate Report screen under Items make sure that the Tabular Results check box is marked Click the icon next to the Tabular Results item The Tabular Results Properties dialog box opens Next to Include Target Information select Yes to include the target information as shown in the table on the dialog box with the tabular results or select No to exclude the target information Under Tabular Results mark the check boxes for the pieces of information that you want to include as columns in the tabula
186. iment Click OK in the Experiments Selection Window The window closes and the experiments that you added are now listed in the Experiment Area under Project To remove experiments from an existing project 1 2 Open the project to any screen In the Experiment Area under Project locate the experiment that you want to remove from the project and click the adjacent Delete icon Agilent AriaMx Real Time PCR Program Creating and Setting Up an MEA Project 12 A message box opens asking you to confirm that you want to remove the selected experiment 3 Click Yes in the message box to continue The program removes the experiment from the project Include or exclude experiments in the project analysis The program allows you to quickly select which experiments in the project are included in the program s analysis of the project This feature allows you to view the effects of excluding one or more experiments without completely removing those experiments from the project To include or exclude experiments in the analysis 1 Open the project to any screen 2 Inthe Experiment Area under Project clear the check box next to any experiments that you want to exclude from the analysis and mark the check boxes for those experiments that you want included in the analysis The program re analyzes the project data and updates the results accordingly Agilent AriaMx Real Time PCR Program 243 12 Creating and Setting Up an MEA
187. iment 50 Convert an experiment to a new experiment type 51 5 Selecting an Experiment Type 53 Overview of Experiment Types 54 Quantitative PCR 54 Comparative Quantitation 54 Allele Discrimination Fluorescence Probes 55 Allele Discrimination DNA Binding Dye with High Resolution Melt 55 User Defined 55 The Quantitative PCR Experiment Type 56 Multiplexing quantitative PCR experiments 56 Well types for Quantitative PCR experiments 57 The Comparative Quantitation Experiment Type 58 Normalizing chance variations in target levels 58 43 Determining amplification efficiencies for the targets of interest and normalizer targets 59 Including biological replicates in comparative quantitation 60 Well types for Comparative Quantitation experiments 61 4 Agilent AriaMx Real Time PCR Program The Allele Discrimination DNA Binding Dye Experiment Type 62 About HRM calibration plates 62 Well types for Allele Discrimination DNA Binding Dye experiments 63 The Allele Discrimination Fluorescence Probe Experiment Type 64 Well types for Allele Discrimination Fluorescence Probe experiments The User Defined Experiment Type 66 6 Setting Up the Plate 6 7 Overview of the Plate Setup screen 68 The plate map 69 Well type 69 Well name Sample name 69 Target information 70 Replicate number 70 Reference dye 0 The Properties panel 71 Additional tools for setting up a plate 71 Import a plate setup 73 Select and view wells in the plat
188. in Added new user AriaMx1 25 Mar 2014 09 22 01 Admin Password of user AnaMx1 changed 26 Mar 2014 13 29 24 Admin Added new user AriaMx2 26 Mar 2014 13 53 05 Admin Password of user AraM changed 26 Mar 2014 14 43 20 Aria x Invalid username or password Export To Excel Print Agilent AriaMx Real Time PCR Program 309 16 Help for the AriaMx ET Electronic Tracking Software Open the system logs To open the system logs 1 Open the Log Viewer dialog box Admin gt Log Viewer 2 Next to Log select System Logs The table displays list system actions in chronological order Print or export the system logs You can print a copy of the system logs table or export a copy to Microsoft Excel To print the system logs 1 Open the Log Viewer dialog box to the System Logs 2 Click Print The Print dialog box opens 3 Select a printer and click Print The program prints a copy of the table To export the system logs to Excel 1 Open the Log Viewer dialog box to the System Logs 2 Click Export to Excel Excel launches with a copy of the table displayed 3 Save the Excel file if desired 310 Agilent AriaMx Real Time PCR Program Help for the AriaMx ET Electronic Tracking Software 16 Add and remove databases in the AriaMx ET software If you are running the electronic tracking ET version of the AriaMx software and you logged in using an administrator account you can control which primary and archive d
189. inear scale These two commands are part of the Axis Options sub menu Click directly on one of these commands to toggle the orientation of the X and Y axes of the graph When a command has a check mark next to it that indicates that the axis Is oriented in the reverse direction The absence of a check mark indicates that the axis is oriented in the forward ascending direction These two commands are part of the Axis Options sub menu Click directly on one of these commands to turn on or off the AutoScale functionality When turned on this functionality causes the program to automatically set the range of the axis based on the data points present on the graph When a command has a check mark next to it that indicates that AutoScale is on The absence of a check mark indicates that AutoScale is off This command which is part of the Axis Options sub menu opens the Graph Properties dialog box to the Axis Options tab You can customize the scale and range of the axes using the tools on this tab See Customize the graph axes on page 216 Agilent AriaMx Real Time PCR Program 213 10 Viewing Graphical Displays of the Results Legend Options gt Show Legend Legend Options gt Top Bottom Left Right Legend Options gt Edit Legend Grid Options gt Show Both Axis Grid Lines Show X Axis Grid Lines Show Y Axis Grid Lines Hide Grid Lines Grid Options gt Solid Grid Lines Dashed Grid Lines Grid Options gt E
190. ined experiments that include wells with allele designations the Graphical Displays screen includes an Allele Determination graph This graph is useful for viewing the genotype results in Allele Discrimination experiments that use two differentially labeled fluorescent probes to detect the two different alleles Each plotted point on the graph represents the coordinates of either the fluorescence values or Cq values for the two targets The position of the data point on the graph indicates the presence or absence of each allele To view the Allele Determinations in a project Click Graphical Displays in the Experiment Area panel on the left side of the screen If the Allele Determination graph is not already displayed click the icon for this graph at the bottom of the screen ea rg The panel on the right side of the screen has tools for adjusting some of the analysis parameters for the allele determinations These tools are the same as those that are available for the Allele Determination graph for a single experiment See View the Allele Determination graph on page 206 for instructions In a project allele determinations are always compared by experiment The program generates a separate graph for each experiment in the project and analyzes the data from each experiment separately 264 Agilent AriaMx Real Time PCR Program 14 Generating Multiple Experiment Analysis Reports and Exporting Results Generate report of MEA project
191. ing Graphical Displays of the Results 216 The color menu closes and the new color is displayed in the Background Color drop down list Optional Click the Apply To All Plots icon to set the new color as the background for all graphs in the experiment Click Close The dialog box closes and the graph displays the new background color To reset the zoom level of a graph back to 100 1 2 3 Open the Graph Properties dialog box to the General tab Next to Zoom click Reset Click Close The dialog box closes and the zoom level of the graph is reset to 100 You can also reset the zoom level from the short cut menu See Zooming on page 171 for instructions on how to zoom Customize the graph axes The tools on the Axis Options tab of the Graph Properties dialog box allow you to customize the orientations and scales of the X and Y axes To set the scale linear or log of the X or Y axis 1 2 Open the Graph Properties dialog box to the Axis Options tab Next to X Axis Scale or Y Axis Scale select Linear to plot the values on the selected axis in a linear scale or select Log to plot the values on a log scale Click Close The dialog box closes and the program adjusts the graph according to your selection To manually set the upper and lower limits on the scale of an axis 1 2 Open the Graph Properties dialog box to the Axis Options tab Under the heading for the desired axis next to Autoscale sel
192. ing dye such as SYBR Green or EvaGreen dye The thermal profile includes a melt segment so that melt curves of the targets can be generated Even DNA amplicons that differ in sequence by only a single nucleotide will yield slightly different melt curves An Allele Discrimination experiment that uses HRM analysis needs to include positive control samples for each base pair possibility at the SNP location homozygous as well as heterozygous positive control samples On the Graphical Displays screen Difference Plots show the difference in fluorescence between two plots during a melt ramp Y axis as a function of temperature X axis By graphing the difference in fluorescence the program can detect even slight differences between two melt curves You can use the difference plots to compare samples of unknown genotype to positive control samples and visually determine which genotype group the target in the unknown sample falls into About HRM calibration plates During the HRM segment of the thermal profile the instrument ramps the temperature of the thermal block in 0 2 C increments At any given target temperature very slight differences in the exact temperature may exist from well to well across the thermal block The purpose of an HRM calibration plate HCP is to calculate an offset temperature for each well in order to help normalize these temperature variations The offset temperature is the difference between the Tm melting temperature
193. int a copy of the table that appears on the User Settings tab of the User Management dialog box You can also export a copy of the table to Microsoft Excel To print the user account table 1 Open the User Management dialog box to the User Settings tab 2 Click Print The Print dialog box opens 3 Select a printer and click Print The program prints a copy of the table To export the user account table to Excel 1 Open the User Management dialog box to the User Settings tab 2 Click Export to Excel Excel launches with a copy of the user account table displayed 3 Save the Excel file if desired Agilent AriaMx Real Time PCR Program 301 16 Help for the AriaMx ET Electronic Tracking Software Archive and restore experiments in the AriaMx ET software If you are running the electronic tracking ET version of the AriaMx software and you logged in using an administrator account you can archive experiments to an archive database and restore previously archived experiments To open the Archive dialog box At the top of the program window click Admin gt Archive Experiments To open the Restore dialog box At the top of the program window click Admin gt Restore Experiments In order to archive or restore experiments your PC must be running MSDTC service See the AriaMx Setup and User s Guide for instructions on configuring and starting the MSDIC service Archive experiments The Archive dialog box has tools for archiving ex
194. ion definition that is automatically loaded You can also configure your own report and save the definition for later use see Create or edit report configuration definitions on page 270 To load a report configuration definition e In the Report Configuration panel of the Generate Report screen next to Definition select the saved report configuration definition from the drop down list The program updates the report preview and the settings under Items and Header amp Footer according to the selected definition After you load a definition you can still make edits to the report configuration Select the items to include in the report The programs offers a variety of items that you can include in the report Each item takes one or more page in the report The Cover Page and Tabular Results items are customizable Agilent AriaMx Real Time PCR Program 267 14 Generating Multiple Experiment Analysis Reports and Exporting Results 268 To include and exclude item from the report In the Report Configuration Panel of the Generate Report screen under Items mark the check boxes for the items that you want to include in the report Clear the check boxes for any items that you do not want to include The preview of the report in the center of the screen includes only the marked items Above each page is the name of the report item displayed on that page To customize the elements of the Cover Page 1 In the Report Conf
195. ists the users on the database with experiments listed under each user and previous snapshots for the experiment listed below as described in the image below Agilent AriaMx Real Time PCR Program 307 16 Help for the AriaMx ET Electronic Tracking Software Experiment Name Date Username Admin Experiment name Quantitative PCR 10 Fold Example 2 Items h fth Quantitative PCR 10 Fold Example 27 Mar 2014 07 15 09 napsnots of the Quantitative PCR 10 Fold Example 27 Mar 2014 07 16 46 experiment 3 Click on an experiment name or snapshot in the table To help locate a particular experiment type a search term into the Search field at the top of the dialog box The table at the bottom of the dialog box shows all actions taken by the user for a selected snapshot if you selected a snapshot or for the entire experiment if you selected an experiment 308 Time Stamp User Description 27 Mar 2014 07 15 09 Admin Experiment imported by Admin 27 Mar 2014 07 16 46 Admin Analysis Replicate Off 27 Mar 2014 07 16 46 Admin Analysis Background Based Threshold End Cycle Old value 9 New value 10 Print or export the audit trail logs You can print a copy of the table that appears at the bottom of the Log View Audit Trail Logs dialog box You can also export a copy of the table to Microsoft Excel To print the table 1 2 Open the Log Viewer dialog box to the Audit Trail Log
196. it plateaus To edit a plateau temperature 1 In the display click directly on the plateau temperature that you want to edit The temperature becomes an editable field 2 Type the desired temperature into the field or click the buttons until you reach the desired temperature 3 Press Enter or click anywhere outside of the field You can also adjust plateau temperature by clicking on the plateau and dragging it up or down to a new temperature To edit a plateau duration 1 In the display click directly on the plateau duration that you want to edit durations are displayed as minutes seconds The duration becomes an editable field 2 Type the desired duration for the plateau into the field or click the buttons until you reach the desired duration Agilent AriaMx Real Time PCR Program 131 7 132 Setting Up the Thermal Profile 3 When typing in the duration you can either type the number of seconds and let the program convert it to the minutes seconds format e g an entry of 120 would be converted to 2 00 or you can type in the duration using the minutes seconds format Press Enter or click anywhere outside of the field To add a plateau 1 2 On the display locate the segment to which you want to add a plateau Within that segment select the existing plateau that needs to precede the new plateau To select a plateau click directly on it The plateau becomes red in the display
197. ith these terms the warranty terms in the sep arate agreement shall control Technology Licenses The hardware and or software described in this document are furnished under a license and may be used or copied only in accor dance with the terms of such license Restricted Rights Legend U S Government Restricted Rights Soft ware and technical data rights granted to the federal government include only those rights customarily provided to end user cus tomers Agilent provides this customary commercial license in Software and techni cal data pursuant to FAR 12 211 Technical Data and 12 212 Computer Software and for the Department of Defense DFARS 252 227 7015 Technical Data Commercial Items and DFARS 227 7202 3 Rights in Commercial Computer Software or Com puter Software Documentation Safety Notices CAUTION A CAUTION notice denotes a haz ard It calls attention to an operat ing procedure practice or the like that if not correctly performed or adhered to could result in damage to the product or loss of important data Do not proceed beyond a CAUTION notice until the indicated conditions are fully understood and met A WARNING notice denotes a hazard It calls attention to an operating procedure practice or the like that if not correctly per formed or adhered to could result In personal injury or death Do not proceed beyond a WARNING notice until the indicated condi tions are fully understoo
198. ive Standard data from all experiments To compare by target 1 Select the Standard Curve graph on the Graphical Displays screen 2 Inthe right panel next to Compare select Target Consolidate the standard curves When you select to consolidate the standard curves the program displays all of the standard curves from all experiments on a single graph The program calculates the template quantities in the same manner as it does when you compare the data by target To consolidate the standard curves 1 Select the Standard Curve graph on the Graphical Displays screen 2 Inthe right panel next to Compare select Consolidated Agilent AriaMx Real Time PCR Program 261 13 Analyzing Multiple Experiment Analysis Project Results Compare Relative Quantity charts in a project 262 For projects comprised of Comparative Quantitation experiments or User Defined experiments that include a set of Calibrator wells the Graphical Displays screen includes a Relative Quantity chart This chart is a bar graph that shows the amount of target present in the experimental samples the Unknown wells relative to the associated reference sample the Calibrator wells after the program has normalized the quantities using data from the normalizer target To view the Relative Quantity for a project Click Graphical Displays in the Experiment Area panel on the left side of the screen If the Relative Quantity chart is not already displayed click the Relativ
199. ized fluorescence values or the first derivative of those values multiplied by 1 Typically the graphs are plotted using first derivative values multiplied by 1 To select the type of fluorescence data displayed in the Raw Derivative Curve 1 Select the Melt Curve Raw Derivative Curve graph on the Graphical Displays screen 2 In the right panel select one of the options next to Fluorescence Term The possible options are R Raw fluorescence Rn Normalized fluorescence normalized to reference dye R T First derivative of the raw fluorescence multiplied by 1 Rn T First derivative of the normalized fluorescence multiplied by 1 Agilent AriaMx Real Time PCR Program Viewing Graphical Displays of the Results 10 See Select which data collection points to analyze on page 161 for information on specifying which data collection point to use for generating the raw derivative curves Adjust the graph properties You can adjust certain properties of the Melt Curve Raw Derivative Curve graph e g the scales of the axes the graph title and the back ground color through the short cut menu and the Graph Properties dialog box See Customize graph properties on page 211 for more information Specify the use of smoothing You can select to apply a Savitzky Golay curve smoothing algorithm to the raw derivative curves for a reference see Savitzky and Golay Analytical Chemistry 1964 36 8 1627 1639 Smoothin
200. king this button will unselect all wells To select an entire row or column of wells 1 Click the corresponding row header A H or column header 1 12 To select a range of adjacent wells 1 Click and hold the left mouse button as you drag the cursor across the wells to be selected A visible marking rectangle appears 74 Agilent AriaMx Real Time PCR Program Setting Up the Plate 6 2 When all of the required wells are included in the rectangle release the left mouse button The range of wells is selected To select a group of non contiguous wells 1 Press Ctrl as you click individually on each of the wells to be selected Unselect wells in the plate map Use one of the following approaches to unselect wells e To unselect individual wells press Ctrl as you click on the wells to be unselected e To unselect a selected row or column click on the header for that row or column e To rapidly unselect all wells click twice on the button in the upper left hand corner of the plate This will select and then unselect all wells View details of a well or wells When the plate map is displaying all 96 wells details such as target names and standard quantities are not displayed The Plate Setup screen offers multiple ways to view the detailed properties of a single well or multiple wells View details of an individual well To view the detailed properties of an individual well e Hover the cursor over the well The
201. ld type in the desired replicate number for the selected wells or click the buttons to enter the desired number The assigned replicate number appears in each selected well Agilent AriaMx Real Time PCR Program Setting Up the Plate 6 To assign replicates using Auto Increment 1 On the Plate Setup screen assign well types as needed for your experiment 2 Inthe Properties panel under Replicates select Manual if not already selected Replicates Kenti Auto Assign Replicate z Number 6 Auto Increment T a 3 Click Auto Increment When you hover your cursor anywhere on the plate map an icon of the number 1 appears next to the cursor 9 4 With the cursor click and drag across the group of wells that you want to assign as replicate number 1 The program assigns the wells to replicate number 1 and the icon next to the cursor changes to a number 2 5 Click and drag across the group of wells that you want to assign as replicate number 2 The program assigns the wells to replicate number 2 and the icon next to the cursor changes to a number 3 6 Continue assigning replicate numbers for the remainder of the plate When finished click Auto Increment to turn off the Auto Increment function Agilent AriaMx Real Time PCR Program 125 6 126 Setting Up the Plate Assign quantities to Standard wells In order to generate a standard curve from your data you need to assign the initial template quanti
202. le file called a project Project files are given the extension amxp to differentiate them from experiment files which have the extension amad The data from the experiments in a project may be grouped together based on target name or maintained as separate data sets The analysis and display of the data depend on how you set up the experiment plates and how you choose to compare the experiment data Applications MEA in the AriaMx program was designed for the following purposes e In projects comprised of Quantitative PCR experiments you can use multiple experiment analysis to determine the quantity of a particular target in an unknown sample using a standard curve for the target that was generated in a separate experiment This capability allows you to compare unknown sample data from multiple experiments to the same standard curve thereby eliminating the need to run standards with every experiment that includes amplification of that target e In projects comprised of Comparative Quantitation experiments you can normalize the quantity of a target of interest to a normalizer target that was run in a separate experiment The program uses the sample name to associate the target of interest wells with the normalizer wells This capability is particularly useful when you are screening a sample for expression levels of many different target genes and you want to normalize them all to the same normalizer e In all project types you can display
203. llows you to monitor a run by watching the real time changes in fluorescence levels in the individual wells The program displays these changes as raw data plots which are graphs that plot the fluorescence values on the Y axis in relative fluorescence units RFU To monitor the raw data plots 1 Connect to the running instrument 2 Open the Raw Data Plots screen On the left side of the screen is a representation of the experiment plate During the run the raw data are plotted and displayed in each well The Y axis of the plots shows the level of raw fluorescence If monitoring data collection for an Amplification segment the X axis plots the cycle number If monitoring data collection for a melt segment the X axis plots the temperature Agilent AriaMx Real Time PCR Program Running and Monitoring Experiments 8 On the right side of the screen is an expanded view of the raw data plots within a selected well or a set of selected wells Hover your cursor over any individual plot line in this view to see which well and target that line refers to Hover your cursor over a well on the plate to highlight the plot line for that well in the raw data plots Change the display options for the raw data plots Change which data collection marker is used for the plots By default the raw data plots show the data collected during the amplification segment of the thermal profile To select a different data collection marker to display in the raw
204. lls To export instrument data by wells 1 Click Instrument gt Export Instrument Data gt By Wells The Export Instrument Data By Wells dialog box opens 2 Select a folder and file name for the CSV file and click Save The program generates the file and opens it in Microsoft Excel 156 Agilent AriaMx Real Time PCR Program Setting Analysis Criteria Overview of the Analysis Criteria screen 158 Select the wells and well types to include in analysis 159 Select wells for analysis using the plate map 159 Select wells for analysis based on well type 159 Select the targets to include in analysis 160 Select which data collection points to analyze 161 Choose a treatment for replicate wells 162 Assign an HRM calibration plate 163 Assign an HCP for new experiments 163 Assign an HCP using the HCP icon 164 Agg Agilent Technologies 9 Setting Analysis Criteria Overview of the Analysis Criteria screen Your selections on the Analysis Criteria screen determine the settings that the program uses for data analysis To open the Analysis Criteria screen Click Analysis Criteria in the Experiment Area panel on the left side of the screen The Analysis Criteria screen Is only available in post run experiments and in progress experiments that have completed at least one point of data collection The Analysis Criteria screen has an image of the plate map that is based on the settings on the Plate Setup screen At the bottom of the screen are icons th
205. ludes multiple amplification collection points or multiple melt collection points you can specify on the Analysis Criteria screen or on the Graphical Displays screen which data collection point to use for analysis To open the Analysis Criteria screen Click Analysis Criteria in the Experiment Area panel on the left side of the screen To select which data collection point to use for analysis of amplification data 1 On the Analysis Criteria screen hover your cursor over the Data Collection Marker icon at the bottom of the screen Q A window opens displaying the thermal profile with data collection markers Click the data collection marker within an amplification segment that you want to use for analysis of amplification data The window closes and the program uses the selected data collection marker to analyze amplification data To select which data collection point to use for analysis of melt data 1 On the Analysis Criteria screen hover your cursor over the Data Collection Marker icon at the bottom of the screen A window opens displaying the thermal profile with data collection markers Click the data collection marker within a melt segment that you want to use for analysis of melt data The window closes and the program uses the selected data collection marker to analyze melt data Agilent AriaMx Real Time PCR Program 161 9 Setting Analysis Criteria Choose a treatment for replicate wells Data from w
206. m uses a least mean squares curve fitting algorithm to generate the standard curves The panel on the right side of the screen has tools for adjusting some of the analysis parameters To view the Standard Curve Click Graphical Displays in the Experiment Area panel on the left side of the screen If the Standard Curve graph is not already displayed click the Standard Curve icon at the bottom of the screen wa Only the Standard wells that you selected in the Analysis Criteria screen are included in the Standard Curve graph View data for a single data point Each curve on the graph is the plot for a single target in a single Standard well or replicate set The square shaped markers denote the data points from the Standard wells that make up the curve Each of these data points is a fluorescence value typically log scale plotted against the initial template quantity as entered on the Plate Setup screen The program connects these data points to draw the curves displayed on the graph The triangle shaped markers on the graph denote Cq values from Unknown wells To view a summary of the data for a single data point from a Standard well 1 Select the Standard Curve graph on the Graphical Displays screen 2 Hover your cursor over the square marker for the data point of interest A tooltip opens displaying the following information Agilent AriaMx Real Time PCR Program Viewing Graphical Displays of the Results 10 e The well ID re
207. me PCR Program 4 4 Creating Opening an Experiment Open an existing experiment The program allows you to open up to 5 experiments at a time or one project at a time The program displays each open experiment or project on its own tab To open an experiment in a new tab 1 Click the icon to the right of the tabs to open a new tab The new tab opens to the Getting Started screen 2 Click one of the options under Saved e To open an existing experiment that you recently accessed click Recently Opened The center of the screen displays a list of experiments and projects that you have recently opened Double click the experiment you want to open The program opens the experiment to the Plate Setup screen e To browse to the folder of the experiment click Browse The Open dialog box opens Browse to the folder location of the experiment Select the experiment and click Open The dialog box closes and the program opens the experiment to the Plate Setup screen To open an experiment in an already open tab 1 From the toolbar click File gt Open The Open dialog box opens If an experiment or project is currently open in the tab the program closes that experiment or project and prompts you to save any changes 2 Browse to the folder location of the experiment Select the experiment and click Open The dialog box closes and the program opens the experiment to the Plate Setup screen 48 Agilent AriaMx Real Time PCR Program
208. me for the sample Press Enter The sample name appears at the top of the selected wells 4 Repeat steps 2 3 for all samples to be included in the experiment Assign dyes targets Use the check boxes drop down lists and fields under Assign Dyes Targets to indicate which dyes are being used in each well and what target each dye is detecting Dye assignments are required but target name assignments are optional If different wells will be using the same dye to detect different targets assigning a unique name to each target enables the program to treat each target separately during analysis Add Dyes Targets 4 Use Dye Name Target Name O a o C Rox O HEX 0 Gs O os O ser To assign dyes and target names oc 0 0 1 On the Plate Setup screen select all the wells in the plate map that contain the same target For instructions on well selection see Select and view wells in the plate map on page 74 2 Under Add Dyes mark the Use check box for the dye used for target detection in the selected wells Agilent AriaMx Real Time PCR Program 113 6 114 Setting Up the Plate 3 Ifthe fields for entering target names are not displayed click the arrow next to Targets The fields appear to the right of the dye names For the marked dye type a name into the adjacent Target Name field The program assigns the target name to the selected wells If you do not assign a target name the program uses the dye name
209. n e In the Report Configuration panel of the Generate Report screen next to Definition select the saved report configuration definition from the drop down list The program updates the report preview and the settings under Items and Header amp Footer according to the selected definition After you load a definition you can still make edits to the report configuration Select the items to include in the report The programs offers a variety of items that you can include in the report Each item takes one or more page in the report The Cover Page and Tabular Results items are customizable Agilent AriaMx Real Time PCR Program 223 11 224 Generating Reports and Exporting Results To include and exclude item from the report In the Report Configuration Panel of the Generate Report screen under Items mark the check boxes for the items that you want to include in the report Clear the check boxes for any items that you do not want to include The preview of the report in the center of the screen includes only the marked items Above each page is the name of the report item displayed on that page To customize the elements of the Cover Page 1 In the Report Configuration Panel of the Generate Report screen under Items make sure that the Cover Page check box is marked Click the icon next to the Cover Page item The Cover Page Options dialog box opens In the Report Title and Report Description fields edit the c
210. n The saved definition must be for the same experiment type Agilent AriaMx Real Time PCR Program Generating Reports and Exporting Results 11 The program comes preloaded with a default data export definition that is automatically loaded You can also configure a custom definition for later use see Create or edit data export definitions below To load a data export definition e In the Export Configuration panel of the Export Data screen next to Definition select the saved data export definition from the drop down list The program updates the settings in the Export Configuration panel according to the selected definition After you load a definition you can still make edits to the data export configuration Create or edit data export definitions You can save your settings on the Export Data screen as a data export definition The program saves the definitions to your system allowing you to use them again with future experiments of the same type Save changes to the default data export definition as a new definition The program comes preloaded with a default data export definition which is loaded by default for new experiments When the default definition is loaded you can still make edits to the data export settings but you cannot save the changes to the default definition Instead save the settings as a new definition To save changes to the default data export definition as a new definition 1 With the
211. n Replicate Number 6 6 Auto Increment ee 3 Inthe Assign Replicate Number field type in the desired replicate number for the selected wells or click the buttons to enter the desired number The assigned replicate number appears in each selected well To assign replicates using Auto Increment 1 On the Plate Setup screen assign well types as needed for your experiment 2 Inthe Properties panel under Replicates select Manual if not already selected 3 Click Auto Increment When you hover your cursor anywhere on the plate map an icon of the number 1 appears next to the cursor 9 4 With the cursor click and drag across the group of wells that you want to assign as replicate number 1 Agilent AriaMx Real Time PCR Program 109 6 Setting Up the Plate The program assigns the wells to replicate number 1 and the icon next to the cursor changes to a number 2 5 Click and drag across the group of wells that you want to assign as replicate number 2 The program assigns the wells to replicate number 2 and the icon next to the cursor changes to a number 3 6 Continue assigning replicate numbers for the remainder of the plate When finished click Auto Increment to turn off the Auto Increment function 110 Agilent AriaMx Real Time PCR Program Setting Up the Plate 6 Assign plate properties for an Allele Discrimination Fluorescence Probe experiment The controls on Plate Setup screen s Properties panel allow
212. n list These options are not available if you selected to not display grid lines 4 Click Close The dialog box closes and the graph displays the grid color Customize the graph legend The tools on the Legend Options tab of the Graph Properties dialog box allow you to customize the graph s legend including the position of the legend the font size used in the legend text and the color assigned to each plot To show or hide the legend on a graph 1 Open the Graph Properties dialog box to the Legend Options tab 2 Next to Legend select On to display a legend on the graph or select Off to hide the legend 3 Click Close Agilent AriaMx Real Time PCR Program Viewing Graphical Displays of the Results 10 The dialog box closes and the program shows or hides the legend according to your selection To set the position of the legend on a graph 1 Open the Graph Properties dialog box to the Legend Options tab 2 Next to Position select where on the graph you want to display the legend The options are Top Bottom Left and Right These options are not available if you selected to hide the legend 3 Click Close The dialog box closes and the program adjusts the position of the legend according to your selection To adjust the font size of the legend text 1 Open the Graph Properties dialog box to the Legend Options tab 2 Next to Font Size type the desired font size into the field or click the buttons to adjust the v
213. n of the program To lock the program e From the toolbar click File gt Lock The program locks and the Unlock dialog box opens To unlock the program 1 In the Password field of the Unlock dialog box type the password for the logged in user Alternatively if you are an administrator type your administrator username and password into the fields 2 Click Login Log out of the program Logging out of the program logs out the currently logged in user Any user can log back in after the current user logs out To log out of the program e From the toolbar click File gt Logout The program logs out the current user and the Login dialog box opens Agilent AriaMx Real Time PCR Program 291 16 Help for the AriaMx ET Electronic Tracking Software To log in to the program 1 In the Login dialog box type your username and password into the fields 2 Click Login 292 Agilent AriaMx Real Time PCR Program Help for the AriaMx ET Electronic Tracking Software 16 Change your password in the AriaMx ET software By default the program automatically prompts users to reset the password for their account every 90 days However users can change the password for their AriaMx ET account at any time using the instructions provided here Administrators can reset the password for any user account at any time See Change a user s password on page 300 for instructions To change the password for your AriaMx ET user account
214. n order to differentiates between those different targets If you do not assign a target name to a marked dye the program uses the dye name as the target name To assign dyes and target names 1 On the Plate Setup screen select the first experiment for editing 2 Select all the wells in the plate map that contain the same target Agilent AriaMx Real Time PCR Program Creating and Setting Up an MEA Project 12 3 Under Add Dyes if the fields for entering target names are not displayed click the arrow next to Targets The fields appear to the right of the dye names 4 For the dyes that were used to amplify multiple targets across all experiments in the project type a name into the adjacent Target Name field The program assigns the target names to the selected wells 5 Repeat steps 1 4 for all wells and experiments included in the project Make sure to give the same target name to identical targets and different target names to different targets Edit plate properties You can assign plate properties to the experiments of a project in the same way that you assign them for an individual experiment The tools for assigning these properties are located in the panel on the right side of the Plate Setup screen The content of this panel depends on the experiment type See the following help topics for information on your experiment type Assign plate properties for a Quantitative PCR DNA Binding Dye experiment on page 81
215. n save the workbook with the file name and folder location of your choice To configure and export data to an Excel file 1 On the Export Data screen next to File Type select Excel 2 Under Items mark the items that you want to include in the file See Select the items to include in the file on page 228 A preview of the file appears in the center of the screen 3 Click Export Data Microsoft Excel opens to the new workbook 4 In Excel save the file as desired Export to text files When you export data results as a text file the program creates a separate text file for each data item included in the data export configuration The file names include the experiment name and data item The program prompts you to select a folder location for the files You can then open the files in the text editing program of your choice To configure and export data to text files 1 On the Export Data screen next to File Type select Text 2 Under Items mark the items that you want to include in the file See Select the items to include in the file on page 228 3 Click Export Data The Browse For Folder dialog box opens 4 Select the folder where you want to save the text files and click OK The program creates the files and saves them in the designated folder Agilent AriaMx Real Time PCR Program 229 11 230 Generating Reports and Exporting Results Export data to an RDML file MIQE guidelines recommend the Real time
216. nd click Open The dialog box closes and the selected experiment appears in the list on the Getting Started screen Repeat steps 2 3 for all experiments that you want to include in the experiment You can add up to 8 experiments to a project You can also add experiments after you create the project See Add or remove experiments from the project on page 242 In the Project Name field at the bottom of the Getting Started screen type a name for the new project Click Create The program creates the new project and opens the project to the Plate Setup screen Agilent AriaMx Real Time PCR Program Creating and Setting Up an MEA Project 12 Open an existing MEA project In order to open a project the program requires you to close all other experiments and tabs To open a project from the Getting Started screen 1 On the Getting Started screen under Saved click Browse The Open dialog box opens 2 Select the project and click Open The dialog box closes and the program opens the project to the Plate Setup screen You may be prompted to save any open experiments and or close any open tabs To open a project from the File menu 1 Click File gt Open The Open dialog box opens If an experiment or project is currently open in the selected tab the program closes that experiment or project and prompts you to save any changes 2 Select the project and click Open The dialog box closes and the program opens the proje
217. needed for your experiment 2 Inthe Properties panel under Replicates select Manual if not already selected 3 Click Auto Increment When you hover your cursor anywhere on the plate map an icon of the number 1 appears next to the cursor 4 With the cursor click and drag across the group of wells that you want to assign as replicate number 1 Agilent AriaMx Real Time PCR Program 101 6 102 Setting Up the Plate The program assigns the wells to replicate number 1 and the icon next to the cursor changes to a number 2 Click and drag across the group of wells that you want to assign as replicate number 2 The program assigns the wells to replicate number 2 and the icon next to the cursor changes to a number 3 Continue assigning replicate numbers for the remainder of the plate When finished click Auto Increment to turn off the Auto Increment function Assign quantities to Standard wells If your Comparative Quantitation experiment includes running a set of standards in order to calculate amplification efficiencies of your targets you need to assign the initial template quantity to each Standard well Standard Quantities Select Target CYS Starting Amount 0 000e 0 A factor of ix Units for Plate nanograms To assign the standard quantities for a target in the Standard wells 1 On the Plate Setup screen select the Standard wells For instructions on well selection see Select and
218. ng a ramp within a melt or high resolution melt segment you can edit the resolution and the soak time The resolution sets the temperature increments of the ramp The soak time is the length of time that the instrument holds each incremental temperature during the ramp Melt segments can have only one data collection marker You can only change the ramp resolution if the experiment is an Allele Discrimination DNA Binding Dye experiment or a User Defined experiment Agilent AriaMx Real Time PCR Program 135 7 Setting Up the Thermal Profile To change the resolution of a ramp 1 On the melt segment click the area below the segment name that displays the resolution and soak time Melt Resolution 0 5 C Soak Time 10s The Ramp Mode dialog box opens Ramp Mode Settings Resolution C 0 2 ecu Soak Time s 10 Cancel 2 Next to Resolution C select 0 2 to set the ramp to 0 2 C increments or select 0 5 to set the ramp to 0 5 C increments A resolution of 0 2 yields a high resolution melt curve 3 Click OK to close the dialog box and save your changes Melt segments with a resolution of 0 2 are named High Resolution Melt Melt segments with a resolution of 0 5 are named Melt 136 Agilent AriaMx Real Time PCR Program Setting Up the Thermal Profile 7 To change the soak time for a ramp 1 On the melt segment click the area below the segment name that displays the resolution and soak time Re
219. nition as a new definition The program comes preloaded with a default data export definition which is loaded by default for new projects When the default definition is loaded you can still make edits to the data export settings but you cannot save the changes to the default definition Instead save the settings as a new definition To save changes to the default data export definition as a new definition 1 With the default definition loaded on the Export Data screen make your desired changes to the settings 2 Inthe Export Configuration Panel next to Definition expand the drop down list and click Add New The Add New Definition dialog box opens Agilent AriaMx Real Time PCR Program Generating Multiple Experiment Analysis Reports and Exporting Results 14 3 Inthe Definition Name field type a name for the definition or use the name provided 4 Make sure the Use Current Settings check box is marked 5 Click Add The dialog box closes and the program saves the data export configuration to the new definition Save changes to a custom data export definition If you loaded a saved definition other than the default definition and then made changes to the data export settings you can save the changes either by overriding the existing definition or by creating a new definition To save changes to the data export settings by overriding the existing definition 1 After loading the saved definition on the Export Data scre
220. notype Calls select Manual The program adds the rectangles to the graph grouping data points with the same genotype call 208 Agilent AriaMx Real Time PCR Program Viewing Graphical Displays of the Results 10 3 Move the border of the any of the rectangles to include or exclude data points a Hover your cursor over the border that you want to reposition b Click and drag the border to a new position To specify which genotype groups have a rectangle displayed on the graph 1 Select the Allele Determination graph on the Graphical Displays screen 2 Right click on the graph A short cut menu opens The menu includes a list of the possible genotypes Allele A Allele B Both and None The genotypes that are already represented on the graph with a rectangle have a check mark next to them 3 Click on the genotypes in the short cut menu to add or remove rectangles as desired Each time you click a genotype the short cut menu closes and the program updates the rectangles on the graph Re open the short cut menu to make further changes Adjust the graph properties You can adjust certain properties of the Allele Determination graph e g the graph title and the background color through the short cut menu and the Graph Properties dialog box See Customize graph properties on page 211 for more information Adjust the last cycle When you select to display the graph by fluorescence rather than Cq the cycle number enter
221. o Type the well type appears at the top of the selected wells 3 Repeat steps 1 2 for all well types to be included in the experiment Assign well names After you assign wells to a well type you can if desired assign custom well names E Type ES Well Name Agilent AriaMx Real Time PCR Program 81 82 Setting Up the Plate To assign well names 1 On the Properties panel of the Plate Setup screen next to Show select Name The Well Name field becomes available for typing Select all the wells in the plate map that you want to assign to the same well name For instructions on well selection see Select and view wells in the plate map on page 74 You must assign a well to a well type before you can assign it a well name In the Well Name field type the well name for the selected wells Press Enter The Well Name appears at the top of the selected wells Repeat steps 2 3 for all well names that you want to assign Assign dyes targets Use the check boxes drop down lists and fields under Assign Dyes Targets to indicate which dyes are being used in each well and what target each dye is detecting Dye assignments are required but target name assignments are optional If different wells will be using the same dye to detect different targets assigning a unique name to each target enables the program to treat each target separately during analysis Add Dyes Targets 4 Use Dye Name Target Name E Fam
222. o a number 3 6 Continue assigning replicate numbers for the remainder of the plate When finished click Auto Increment to turn off the Auto Increment function Agilent AriaMx Real Time PCR Program 117 Setting Up the Plate Assign plate properties for a User Defined experiment 118 The controls on Plate Setup screen s Properties panel allow you to create a customized plate setup for experiments of the type User Defined To open the Plate Setup screen Click Plate Setup in the Experiment Area panel on the left side of the screen Assign well types Use the Well Types drop down list to assign well types to all the wells used in the experiment To assign well types 1 On the Plate Setup screen select all the wells in the plate map that are of the same type For instructions on well selection see Select and view wells in the plate map on page 74 2 Select a well type from the Well Types drop down list in the Properties panel When the Show setting on the Properties panel is set to Type the well type appears at the top of the selected wells 3 Repeat steps 1 and 2 for all well types to be included in the experiment Assign well names After you assign wells to a well type you can if desired assign custom well names Show Type EALER Sample Well Name Sample Name Agilent AriaMx Real Time PCR Program Setting Up the Plate 6 To assign well names 1 On the Properties panel of the Plate Setup screen n
223. og box the next time a user archives an experiment Remove an AriaMx ET database If you have previously added databases and have multiple databases available on your system you can remove a database Removing a primary database makes it no longer available in the Login dialog box that opens when users launch the AriaMx ET program Removing an archive database makes it no longer available when users archive or restore an experiment Agilent AriaMx Real Time PCR Program 313 16 Help for the AriaMx ET Electronic Tracking Software Fi mr oy Remove Database Reference Database Archive Remove Server Instance Database Name local ARIA2 Ania 1CFRPart11 C CHUIS7OGKXARIA2 Anak 1CFRPart11 Remove Cancel To remove a database 1 2 314 Open the Remove Database dialog box Admin gt Remove Database Next to Database select Primary to remove a primary database or Archive to remove an archive database The program populates the table with all available primary databases if Primary is selected or all available archive databases if Archive is selected In the table mark the check box in the Remove column for the database that you want to remove Note that you cannot mark the check box for the currently logged in database the row is grayed out in the table Click Remove The database is removed from the table Agilent AriaMx Real Time PCR Program Help for the AriaMx ET Electronic Tracking
224. on Select wells for analysis based on well type You can select specific wells to include in the analysis based on well type To select specific well types 1 On the Analysis Criteria screen hover your cursor over the Display Well Types icon at the bottom of the screen aoe ooo oon A window opens showing all well types in use on the plate 2 For any well types that you do not want to include clear the check box next to the well type name You can remark the check box at any time to reselect those wells Agilent AriaMx Real Time PCR Program 159 9 Setting Analysis Criteria Select the targets to include in analysis From the Analysis Criteria screen or from the Graphical Displays screen you can select which targets to include in the results displayed on the Graphical Displays screen To open the Analysis Criteria screen Click Analysis Criteria in the Experiment Area panel on the left side of the screen To select specific targets 1 On the Analysis Criteria screen hover your cursor over the Display Targets icon at the bottom of the screen A window opens showing all targets in use on the plate 2 For any targets that you do not want included in the analysis clear the check box next to the target name You can remark the check box at any time to reselect those targets 160 Agilent AriaMx Real Time PCR Program Setting Analysis Criteria 9 Select which data collection points to analyze If your thermal profile inc
225. on occurrence in a well The default is 99 Multicomponent Multicomponent is a term used for distinguishing the contribution that each dye and the background makes to the total fluorescence spectra detected Agilent AriaMx Real Time PCR Program 323 17 Reference Help and Troubleshooting amp Support Trademarks Excel Microsoft and PowerPoint are registered trademarks of Microsoft Corporation SYBR is a registered trademark of Molecular Probes Inc 324 Agilent AriaMx Real Time PCR Program Reference Help and Troubleshooting amp Support 17 Troubleshooting and Support Troubleshooting Guide Observation Unable to install the AriaMx software Very low amplification signal Decreased volume in sample containers at end of run Unexpected results in one sample Suggestion You will be unable to install the software if the account that was used to log in to the PC does not have administrative rights Log in under an account with admin rights If the computer is on a network you may have to contact your IT department Low amplification signal could indicate a possible problem with the probe or the stock of dye that was used A good indication of this type of problem would be if the reference dye is showing good signal but the fluorophore Is not If SYBR Green is being used verify the concentration If using a new probe or a new lot of a probe the problem could be the fluorophore You can test the probe by perf
226. on experiment on page 95 Assign plate properties for an Allele Discrimination DNA Binding Dye experiment on page 104 Assign plate properties for an Allele Discrimination Fluorescence Probe experiment on page 111 Assign plate properties for a User Defined experiment on page 118 To hide the Properties panel click the arrow icon in the upper left corner of the panel Click the arrow again to display the Properties panel Additional tools for setting up a plate Copy Paste To copy well information first select the wells that contain the information to be copied Then press Ctrl c or right click on the plate map and click Copy to copy the properties of the selected wells to the clipboard You can later paste the properties into other wells within the same experiment or in another open experiment Agilent AriaMx Real Time PCR Program 71 6 72 Setting Up the Plate To paste well information from the clipboard to a set of wells first select the wells that you want to paste into Then press Ctrl v or right click on the plate map and click Paste to paste the well information into a selected set of wells You can paste into wells within the same experiment or into wells of an experiment that is open in another tab of the program provided that the properties of the wells are compatible with the experiment type See Select and view wells in the plate map on page 74 for instructions on selecting sub s
227. on the Adjust button indicates that the baseline settings have already been manually adjusted The Baseline Correction dialog box opens Agilent AriaMx Real Time PCR Program 175 10 Viewing Graphical Displays of the Results J Baseline Correction Baseline Cycle range for each displayed target indicates all targets whose cycles differ from the calculated values Start Cycle End Cycle fl E g T 5 4 ee Start Cycle 4 B G26 Select au Reset i Cancel To adjust the baseline cycle range 1 Open the Baseline Correction dialog box 2 Select the plots a plot is one target in one well that you want to reset to the adaptive values by clicking the appropriate row in the table Press Ctrl while clicking to select more than one plot Click Select All to select all plots in the table 3 Inthe Start Cycle field at the bottom of the dialog box type the cycle number that will be the first cycle in the baseline cycle range or click the buttons to adjust the value in the field to the desired cycle number 176 Agilent AriaMx Real Time PCR Program Viewing Graphical Displays of the Results 10 4 In the End Cycle field type the cycle number that will be the last cycle in the baseline cycle range or click the buttons to adjust the value in the field to the desired cycle number 5 Click Apply to apply this baseline cycle range to the selected plots The program updates the table to show the n
228. on the settings on the Plate Setup screen At the bottom of the screen are icons that provide access to menus for making selections on which data you want included in the results displayed on the Graphical Displays screen Toggle display between one experiment and all experiments In a project the Analysis Criteria screen and Graphical Displays screen includes a toggle button for switching between two distinct modes as described in the table below When the toggle button looks like this the program displays the results for all experiments included in the project unless the check box for the experiment is not marked in the Experiment Area panel In this mode the Graphical Displays screen shows only one type of graph at a time e g all the Amplification Plots graphs When the toggle button looks like this the program displays the results for one experiment at a time The program displays results for whichever experiment is selected in the Experiment Area panel Select the wells and well types to include in analysis In a project the method for selecting the wells and well types for analysis is Similar to that used for an individual experiment See Select wells in the plate map on page 74 for detailed instructions 248 Agilent AriaMx Real Time PCR Program Analyzing Multiple Experiment Analysis Project Results 13 Select the targets to include in analysis To select specific targets 1 On the Analysis Criteria screen hove
229. ons You can save your custom report configuration as a report configuration definition You can then load the saved definition into other projects of the same experiment type Save changes to the default report configuration definition as a new definition The program comes preloaded with a default report configuration definition which is loaded by default for new projects When the default definition is loaded you can still make edits to the report configuration but you cannot save the changes to the default definition Instead save the report configuration as a new definition To save changes to the default definition as a new definition 1 With the default definition loaded on the Generate Report screen make your desired changes to the report configuration 2 Inthe Report Configuration Panel next to Definition expand the drop down list and click Add New The Add New Definition dialog box opens Agilent AriaMx Real Time PCR Program Generating Multiple Experiment Analysis Reports and Exporting Results 14 3 Inthe Definition Name field type a name for the definition or use the name provided 4 Make sure the Use Current Settings check box is marked Click Add The dialog box closes and the program saves the current report configuration to the new definition Save changes to a custom report configuration definition If you loaded a saved definition other than the default definition and then made changes to the report
230. ontent as desired The title and description are printed on the cover page Under Experiment Information and Other Items mark the check boxes for the pieces of information that you want to include on the cover page Clear the check box for any pieces of information that you do not want to include In the fields labeled Left Center and Right type the text that you want to appear at the bottom of the cover page on the left side center and right side Click OK in the Cover Page Options dialog box The dialog box closes and the Cover Page displayed in the report preview includes your changes To customize the information included in the Tabular Results 1 In the Report Configuration Panel of the Generate Report screen under Items make sure that the Tabular Results check box is marked Click the icon next to the Tabular Results item The Tabular Results Properties dialog box opens Next to Include Target Information select Yes to include the target information as shown in the table on the dialog box with the tabular results or select No to exclude the target information Under Tabular Results mark the check boxes for the pieces of information that you want to include as columns in the tabular results Agilent AriaMx Real Time PCR Program Generating Reports and Exporting Results 11 Clear the check box for any pieces of information that you do not want to include The table to the left of the check boxes displays
231. orming a nuclease digestion to ensure it is unquenching as expected incubate 100 nM of probe in 25 uL of 1x buffer with 10 U of DNase or 1 nuclease at room temperature for 30 minutes This should result in a fluorescence level increase of gt 5000 RFU If another probe or a stock of the dye is available you can also try testing that for comparison If the probe or dye stock has been stored in a way that might have exposed it to light low signal could also be due to photobleaching of the fluorophore If the samples are still available you can also run these on a gel to make sure you are actually getting amplified product If not assay optimization or new reagents may be necessary Sample containers are not vapor tight Ensure caps plates are tightly sealed and containers are not malformed Also ensure that proper plasticware was used Check the sample container for contaminating material Check optical clarity of sample container cap Also check to make sure the liquid inside that sample tube did not evaporate during the run which would indicate a vapor leak in that particular sample Vapor leaks can occur if the sample was not sealed properly or incorrect plasticware was used Agilent AriaMx Real Time PCR Program 325 17 Reference Help and Troubleshooting amp Support No amplification plots are visible in This could occur if the wrong data collection point was assigned to the the Graphical Displays screen amplification plots Rese
232. ort data results to an Excel text or RDML file 228 Configure the file and export data 228 Load a saved data export definition 230 Create or edit data export definitions 231 fhe Agilent Technologies 11 Generating Reports and Exporting Results Generate report of results 222 The Generate Report screen allows you to set up preview and create a PDF or PowerPoint report for a post run experiment The content and format of the report are highly customizable and you can save a report configuration for use with additional experiments later on To open the Generate Report screen Click Generate Report in the Experiment Area panel on the left side of the screen View a preview of the report The center of the Generate Report screen displays a page by page preview of what the report will look like with the current configuration settings Above each page is the name of the report item displayed on that page To view all pages of the report preview scroll down To adjust the display size of the pages click the buttons at the bottom of the screen Select report type The report can be a PDF or PowerPoint file To select the report type e In the Report Configuration panel of the Generate Report screen next to Report Type select PDF to select a PDF report or select PowerPoint to select a PowerPoint report Generate the report After you configure the report as desired see the tasks under Configure the report
233. ots graphs If you added a check mark to specific wells or replicate sets using the Check Item command or the Check column in the Results Table use these commands to assign the checked items to a genotype Homozygous A Homozygous B or Heterozygous or to remove genotype calls from the checked items Clear Call command This command is used for manual genotype calling in the Melt Curve Difference Plots graphs If you added a check mark to specific wells or replicate sets using the Check Item command or the Check column in the Results Table use this command to remove genotype calls from the checked items This command is used for manual genotype calling in the Melt Curve Difference Plots graphs It opens the HRM Manual Call Settings dialog box which allows you to assign a color and name to each genotype for use in the difference plots This set of four options is only available for Allele Determination graphs Use the options to specify which genotype groups on the graph are visually grouped together with a rectangle See Display genotype groups on the graph on page 208 for more information These two commands are part of the Axis Options sub menu Click directly on one of these commands to toggle between a linear scale and a log scale for the X and Y axes of the graph When a command has a check mark next to it that indicates that the axis is set to a log scale The absence of a check mark indicates that the axis is set to a l
234. own list 2 Click Save As The Add New Definition dialog box opens 3 In the Definition Name field type a name for the definition or use the name provided 4 Click Add The dialog box closes and the program saves the data export settings to the new definition 232 Agilent AriaMx Real Time PCR Program 12 Creating and Setting Up an MEA Project Quick Start Protocol 234 Overview of multiple experiment analysis 235 Applications 235 Restrictions 236 Guidelines for Comparing Cq Values Across Experiments 237 Reducing plate to plate variability 237 Selecting a method for setting threshold fluorescence levels 238 Create an MEA project 240 Open an existing MEA project 241 Select experiments for a project 242 Add or remove experiments from the project 242 Include or exclude experiments in the project analysis 243 Edit the plate setup of experiments in a project 244 Select an experiment to edit 244 Differentiate between targets across experiments 244 Edit plate properties 245 View the thermal profiles of experiments in a project 246 Agg Agilent Technologies 12 Creating and Setting Up an MEA Project Quick Start Protocol 234 How to create set up analyze and generate reports for a multiple experiment analysis project In the AriaMx program a multiple experiment analysis file is referred to as a project A project consists of one or more post run experiment files Project files have the extension amxp 1 Create the proj
235. p e Allele A the group that is homozygous for allele A e Allele B the group that is homozygous for allele B e Hetero the group that is heterozygous for alleles A and B 210 Agilent AriaMx Real Time PCR Program Viewing Graphical Displays of the Results 10 Customize graph properties You can customize many of the properties of the graphs on the Graphical Displays screen Customization options are available through a short cut menu and on the Graph Properties dialog box Customize graph properties using the short cut menu To open the graphs short cut menu From the Graphical Displays screen right click on the graph whose properties you want to edit The commands available through the graphs short cut menu are described in the table below Reset Zoom If you zoomed in on a graph this command resets the zoom level of the graph back to 100 You can also reset the zoom from the Graph Properties dialog box See Zooming on page 1 1 for instructions on how to zoom Add Marker at X Axis These two commands are only available for Melt Curve graphs Difference Plots Add Marker at Y Axis and Raw Derivative Curve The commands add a vertical marker X axis marker or a horizontal marker Y axis marker to the graph Check Item This command is used for manual genotype calling in the Melt Curve Difference Plots graphs It is only available when you right click on an Unknown well or replicate set in the Results Table The command ad
236. p that contain the same target For instructions on well selection see Select and view wells in the plate map on page 74 2 Under Add Dyes mark the Use check boxes for the dyes used for target detection in the selected wells 3 Ifthe fields for entering target names are not displayed click the arrow next to Targets The fields appear to the right of the dye names 4 For the marked dyes type a name into the adjacent Target Name field The program assigns the target names to the selected wells If you do not assign a target name to a marked dye the program uses the dye name as the target name 5 Repeat steps 1 4 for all wells included in use in the experiment 6 Optional Select a new color to associate with a target a Click the colored dot to right of the Target Name field b In the selection box that opens click the desired color or click Advanced for more color options Select a reference dye You can include a reference dye e g ROX dye to normalize the fluorescence signal of the reporter dye To assign a reference dye e On the Plate Setup screen select the reference dye from the Reference Dye drop down list in the Properties panel The program assigns the target name REF to all wells in use in the experiment and displays an R in the wells of the plate map to indicate that the well contains a reference dye 90 Agilent AriaMx Real Time PCR Program Setting Up the Plate 6 Assign replicates
237. pe 69 Well name Sample name 69 Target information 70 Replicate number 70 Reference dye 70 The Properties panel 71 Additional tools for setting up a plate 71 Import a plate setup 73 Select and view wells in the plate map 74 Select wells in the plate map 74 Unselect wells in the plate map 75 View details of a well or wells 75 Export the plate map image 80 Assign plate properties for a Quantitative PCR DNA Binding Dye experiment 81 Assign plate properties for a Quantitative PCR Fluorescence Probe experiment 88 Assign plate properties for a Comparative Quantitation experiment 95 Assign plate properties for an Allele Discrimination DNA Binding Dye experiment 104 Assign plate properties for an Allele Discrimination Fluorescence Probe experiment 111 Assign plate properties for a User Defined experiment 118 Agg Agilent Technologies 6 Setting Up the Plate Overview of the Plate Setup screen The Plate Setup screen has tools for assigning properties to the wells of the plate so that the program can properly analyze your data To open the Plate Setup screen With an experiment or project file open click Plate Setup in the Experiment Area panel on the left side of the screen File Instrument Experiment Area JS setup Plate Setup Thermal Profile Run pir b Felice bes ir i P ee M E E BERGEEE UOH s A am 68 Agilent AriaMx Real Time PCR Program Setting Up the Plate 6 The plate map
238. periments you cannot transfer data from the instrument to your PC through the network See Retrieve data using a USB drive for instructions Assign an HCP for new experiments The first time you attempt to open the Analysis Criteria or Graphical Displays screen for a new post run experiment that includes an HRM segment the following message box opens on your screen Agilent AriaMx x O Please import an HRM Calibration Plate experiment file in order to perform full HRM analysis _ Import Cancel To assign an HCP from the message box 1 In the message box click Import The Open dialog box opens 2 Browse to the HCP file that you want to assign Select the file and click Open Agilent AriaMx Real Time PCR Program 163 9 164 Setting Analysis Criteria The program assigns the HCP to the experiment and calibrates the melt data accordingly If you click Cancel in the message box shown above you can still assign an HCP using the HCP icon on the Analysis Criteria or Graphical Displays screen See instructions below Assign an HCP using the HCP icon The HCP icon is available on the Analysis Criteria and Graphical Displays screens for all experiments that include an HRM segment To assign an HCP 1 On the Analysis Criteria or Graphical Displays screen click the HCP icon at the bottom of the screen CE The Open dialog box opens Browse to the HCP file that you want to assign Select the file and click Open
239. periments and creating new archive databases Archiving an experiment requires moving it from the primary database to an archive database Once archived users cannot work in an experiment unless an administrator restores it 302 Agilent AriaMx Real Time PCR Program Help for the AriaMx ET Electronic Tracking Software 16 J Archive Select Archive Database Ariatarchive Search Archive Experiment Name Last Modified new expt 2 Mar 2014 13 34 41 Quantitative PCR 10 Fold Example 2 Mar 2014 07 16 46 Quant PCR_SYBR 26 Mar 2014 14 43 04 Comparative Quantitation Multiplex Example copy 1 26 Mar 2014 14 37 36 Comparative Quantitation Multiplex Example copy 6 Mar 2014 14 36 39 J Select All Experiments to Archive Cancel Create an archive database Before you archive an experiment you may wish to create a new archive database Creating an archive database makes it available in the Select Archive Database drop down list in the Archive dialog box To create an archive database 1 Open the Archive dialog box Admin gt Archive Experiments 2 Inthe Select Archive Database drop down list click Create new The Create Archive Database dialog box opens In the Server Name drop down list select the appropriate server In the Password field type your SQL Server password In the Database name field type a name for the new archive database Click Create a oi Ww A message box opens confirming the creation of the
240. plicate number target name and well type or sample name for the plot e The initial template quantity as entered on the Plate Setup screen i e the X axis coordinate e The quantification cycle for the plot i e the Y axis coordinate To view a summary of the data for a single data point from an Unknown well 1 Select the Standard Curve graph on the Graphical Displays screen 2 Hover your cursor over the triangle marker for the data point of interest A tooltip opens displaying the following information e The well ID replicate number target name and well type or sample name for the plot e The initial template quantity as calculated by the program i e the X axis coordinate e The quantification cycle for the plot i e the Y axis coordinate Select a fluorescence data type For the Y axis of the amplification plots you can use baseline corrected fluorescence values with or without normalization to a reference dye To select the type of fluorescence data displayed in the Standard Curve graph 1 Select the Standard Curve graph on the Graphical Displays screen 2 In the right panel select one of the options next to Fluorescence Term The possible options are AR baseline corrected raw fluorescence ARn baseline corrected normalized fluorescence See Select which data collection points to analyze on page 161 for information on specifying which data collection point to use for generating the standa
241. portnumber 144 View information about an instrument 144 Start stop or pause a run 146 Startarun 146 Cancelarun 147 Pausearun 148 Monitorarun 149 Connect to the running instrument 149 Monitor a run by viewing its progress through the thermal profile 150 Monitor a run by viewing the raw data plots 150 Change the display options for the raw data plots 151 Stop monitoring a run 153 Retrieve run data from the instrument 154 Retrieve data through the network 154 Retrieve data using a USB drive 155 Export instrument data toa CSV file 156 Export instrument data by column 156 Export instrument data by target 156 Export instrument data by wells 156 Agg Agilent Technologies 8 Running and Monitoring Experiments Overview of the Instrument Explorer dialog box The Instrument Explorer dialog box has tools for performing a variety of functions that require connecting to an instrument through a local network To open the Instrument Explorer dialog box e For starting and monitoring a run open the Thermal Profile Run Status or Raw Data Plots screen and click Run e For other Instrument Explorer functions at the top of the program window click Instrument gt Instrument Explorer Starting a run is not enabled when you open the dialog box from the Instrument menu However opening the dialog box from the Instrument menu does allow you to monitor a run that has already been started See the following help topics for instructions on specific
242. program searches your local machine and network to identify available servers In the Server Name drop down list select the appropriate server Type your SQL Server password into the Password field Click Connect Next to Database make sure the Primary option is selected In the Select Database drop down select the desired database This drop down lists all primary databases available on the server Agilent AriaMx Real Time PCR Program Help for the AriaMx ET Electronic Tracking Software 16 7 Click Add The dialog box closes and the database is listed in the Login dialog box the next time a user launches the program Add an archive database When you archive an experiment the Archive dialog box includes a drop down list of available archive database You can use the tools on the Add Database dialog box to add an archive database to that drop down list To add an archive database 1 Open the Add Database dialog box Admin gt Add Database The program searches your local machine and network to identify available servers In the Server Name drop down list select the appropriate server Type your SQL Server password into the Password field Click Connect Next to Database select Archive a oi A GW N In the Select Database drop down select the desired database This drop down lists all archive databases available on the server 7 Click Add The dialog box closes and the database is listed in the Archive dial
243. r results Agilent AriaMx Real Time PCR Program Generating Multiple Experiment Analysis Reports and Exporting Results 14 Clear the check box for any pieces of information that you do not want to include The table to the left of the check boxes displays a preview of the tabular results based on which columns you select to include To quickly mark all check boxes click Select All To restore the default selections click Restore Defaults 5 Click OK in the Tabular Results Properties dialog box The dialog box closes and the Tabular Results pages displayed in the report preview include your changes To edit the Project Notes 1 Inthe Report Configuration Panel of the Generate Report screen under Items make sure that the Project Notes check box is marked 2 Click the icon next to the Project Notes item The Project Notes text box opens 3 In the text box type any notes that you want to add to the project and include in the report 4 Click Save to save your changes and close the text box To show analysis settings 1 Inthe Report Configuration Panel of the Generate Report screen next to Show Analysis Settings select Yes In the report the analysis settings for each graph are displayed below the graph Select the contents of the header and footer You can select which pieces of information you want to include in the report s header and footer To select the contents of the header and footer e In the Report
244. r to be assigned to the selected wells Press Enter The program adds the biological replicate ID number to the end of the sample name at the top of the selected well s Repeat steps 2 3 for the remaining wells that require a biological replicate ID assignment Agilent AriaMx Real Time PCR Program Setting Up the Plate 6 Assign dyes targets Use the check boxes drop down lists and fields under Assign Dyes Targets to indicate which dyes are being used in each well and what target each dye is detecting Dye assignments are required but target name assignments are optional If different wells will be using the same dye to detect different targets assigning a unique name to each target enables the program to treat each target separately during analysis Add Dyes Targets 4 Use Dye Name Target Name Q Fam s O Rox 0 O os 0O cvs Q ser To assign dyes and target names ooo 0 0 1 On the Plate Setup screen select all the wells in the plate map that contain the same target For instructions on well selection see Select and view wells in the plate map on page 74 2 Under Add Dyes mark the Use check box for the dye used for target detection in the selected wells 3 Ifthe fields for entering target names are not displayed click the arrow next to Targets The fields appear to the right of the dye names 4 For the marked dye type a name into the adjacent Target Name field The program assigns the targ
245. r your cursor over the Display Targets icon at the bottom of the screen A window opens showing all targets in use on the plate 2 For any targets that you do not want included in the analysis clear the check box next to the target name You can remark the check box at any time to reselect those targets Select which data collection points to analyze One experiment displayed If you are displaying only one experiment in the project see Toggle display between one experiment and all experiments on page 248 use the instructions below to select a data collection point for the displayed experiment 1 Hover your cursor over the Data Collection Marker icon at the bottom of the screen A window opens displaying the thermal profile with data collection markers 2 Click the data collection marker that you want to use for analysis All experiments displayed If you are displaying all experiments in the project by default the program uses the data collection point for each experiment that was selected in the experiment at the time the project was created You cannot change the selected collection point unless you toggle to displaying one experiment and then change which data collection marker is selected in the displayed experiment Agilent AriaMx Real Time PCR Program 249 13 Analyzing Multiple Experiment Analysis Project Results Choose a treatment for replicate wells To specify that replicates be treated individually
246. raph The program calculates the Tm values in the same manner as it does when you compare the data by experiment or by target To consolidate the melt curves 1 Select the Melt Curve Raw Derivative Curve graph on the Graphical Displays screen 2 Inthe right panel next to Compare select Consolidated Agilent AriaMx Real Time PCR Program 259 13 Analyzing Multiple Experiment Analysis Project Results Compare standard curves in a project 260 For projects comprised of Quantitative PCR experiments Comparative Quantitation experiments or User Defined experiments that include a set of Standard wells the Graphical Displays screen includes a Standard Curve graph The Standard Curve graph shows a plot of the Cq Y axis versus the log of the initial template quantity in the Standard wells The graph also plots the Cq values from the Unknown wells The program uses a least mean squares curve fitting algorithm to generate the standard curves The panel on the right side of the screen has tools for adjusting some of the analysis parameters To view the Standard Curve for a project Click Graphical Displays in the Experiment Area panel on the left side of the screen If the Standard Curve graph is not already displayed click the Standard Curve icon at the bottom of the screen The panel on the right side of the screen has tools for adjusting some of the analysis parameters for the standard curves These tools are the same as those
247. ration plates on page 62 for more information Prepare the plate Use the Agilent HRM Calibration Plate Agilent offers a HRM Calibration Plate that you can use for HRM calibration Agilent part number 5190 7701 The plate comes pre aliquoted with a master mix containing EvaGreen dye See the plates s user manual for more information Visit www agilent com genomics for ordering information on the HRM Calibration Plate Prepare the plate with your own reagents Alternatively you can prepare your own reagent mixture to be used in the HCP run The 1x mixture needs to contain a purified amplicon at a concentration of 0 1 0 3 mM Ideally the amplicon has a melting temperature Tm close to that of the amplicon that you will be analyzing in your Allele Discrimination experiment The 1x mixture also needs to contain the same DNA binding dye at the same concentration that you use in the QPCR reactions for the Allele Discrimination experiment Agilent recommends using the 2x master mix from the Agilent HRM Calibration Plate Kit to prepare your reagent mixture Instead of using a purified amplicon in your reagent mixture you can use a template primers and polymerase to produce the amplicon during the HCP run If you choose this approach you need to add an amplification segment to the default HCP thermal profile Agilent AriaMx Real Time PCR Program 35 3 36 Performing Hardware and Software Tests and HRM Calibrations Note that
248. rd curve Agilent AriaMx Real Time PCR Program 197 10 Viewing Graphical Displays of the Results 198 View the R squared values slopes and amplification efficiencies The Target Information Table displays information about each target on the Standard Curve graph This information includes the amplification efficiency which is calculated from the slope the R squared R3 value the slope of the standard curve plot and the point where it intercepts the Y axis Target Information Table Efficiency Intercept 94 41 464 20 54 92 1 20 48 39 39 20 77 935 53 21 26 33 92 20 9 The R value is an indicator of the quality of the fit of the standard curve to the standard data points plotted The value is always between 0 and 1 and the closer the value is to 1 the better the fit of the line The slope of the curve is directly related to the average amplification efficiency throughout the cycling reaction The program uses the following equation to calculate slope y m log x b where m is the slope of the line PCR efficiency is the percentage of template molecules that are doubled every cycle The equation that relates the slope to amplification efficiency is PCR efficiency 10 1 slope 1 Based on this equation a PCR reaction with 100 efficiency results in a standard curve with a slope of 3 322 To view the Target Information Table 1 Select the Standard Curve graph on the Graphical Displays screen The table i
249. reates a new presentation that contains the image of the selected graph Send to Excel This command launches Microsoft Excel and creates a new spreadsheet that contains the data from the selected graph Restore Default Settings This command sets all the graph properties back to their default settings Customize graph properties using the Graph Properties dialog box To open the Graph Properties dialog box From the Graphical Displays screen double click on the graph whose properties you want to edit Customize general graph properties The tools on the General tab of the Graph Properties dialog box allow you to set many of the general properties for the graph including title and background color To edit a graph title 1 Open the Graph Properties dialog box to the General tab 2 Inthe Graph Title field type the desired title for the graph 3 Click Close The dialog box closes and the graph displays the new title To customize the background color of a graph 1 Open the Graph Properties dialog box to the General tab 2 Expand the Background Color drop down list to view a menu of standard background colors 3 Select a background color e If the standard menu includes your desired color click directly on it e If you need a color not included on the palette click Advanced to view an advanced menu for color selection Use the color picker tools to create a custom color Agilent AriaMx Real Time PCR Program 215 10 View
250. rection or calibration factors Quantitate the initial template quantity of a DNA target based ona standard curve Generate and display normalized relative quantity values on a log 2 fold change chart to assess the effects of an experimental treatment on gene expression levels Export any data set directly to Microsoft Excel or to a text file View and analyze the data from several experiments together in a single project using the multiple experiment analysis functions Use high resolution melt analysis for SNP genotyping in an Allele Discrimination experiment Agilent AriaMx Real Time PCR Program Getting Started with the AriaMx Program 1 Getting Started with the AriaMx Software Getting Started with the AriaMx Software Introduction to the AriaMx software The software s Home screen provides an introduction to the program and a list of program features To open the Home screen Click the Home icon near the upper right corner of the program window NOTE If you want the program to open to the Home screen mark the check box near the bottom of the screen labeled Show Home on StartUp Analyze monitor and iew all your samples Agilent Technologies building upon Stratagene s legacy of excellence provides a total solution approach with the AriaMx Real Time PCR System simplifying the challenges you face in experimental setup and data analysis for real time quantitative PCR experiments Whether you are new to or exper
251. reen next to Show select Name The Well Name field becomes available for typing 2 Select all the wells in the plate map that you want to assign to the same well name For instructions on well selection see Select and view wells in the plate map on page 74 You must assign a well to a well type before you can assign it a well name 3 In the Well Name field type the well name for the selected wells Press Enter The Well Name appears at the top of the selected wells 4 Repeat steps 2 3 for all well names that you want to assign Assign sample names and biological replicates Once well types have been assigned you can specify the sample in each well by assigning sample names Each unique template sample included in the experiment can be assigned its own sample name If the normalizer target and the target of interest are being amplified in different wells be sure to assign the same sample name to these wells so the data are normalized properly during analysis For samples that are biological replicates assign the same sample name to the wells but give the wells different biological replicate ID numbers to keep them differentiated Biological replicates are template samples that were isolated independently but from biologically identical sources see Including biological replicates in comparative quantitation on page 60 for more information on biological replicates Agilent AriaMx Real Time PCR Program Setting
252. required for the fluorescence signal to be detectable above background The AriaMx program offers both automatic and manual methods for determination of the threshold fluorescence level that is used to determine Cq values Typical Quantitative PCR experiments use a standard curve to quantitate the amount of target present in an experimental sample called the unknown sample since the quantity of the target is unknown In this method you set up the plate to amplify a series of standards to generate a standard curve that relates initial template quantity to Cq You generate the standards by serial dilution of a template sample that contains a known quantity of the target under investigation The program then uses the standard curve to derive the initial template quantity of this target in the unknown samples based on their Cq values Multiplexing quantitative PCR experiments When using the AriaMx system the instrument records fluorescence readings for each sample on all five optics modules This allows you to use multiplex PCR for quantitation of multiple targets in the same well by using spectrally distinct dyes to detect each target The AriaMx program reports each target in each well separately on amplification plots and other graphical results displays Agilent AriaMx Real Time PCR Program Selecting an Experiment Type 5 Well types for Quantitative PCR experiments Well Type Description Unknown Contains a complete reaction mixt
253. riaMx Real Time PCR Program Assign plate properties for a User Defined experiment 118 Assign welltypes 118 Assign well names 118 Assign sample names and biological replicates 119 Assign dyes targets 121 Selectareferencedye 122 Designate the normalizer 122 Assign Alleles 123 Assign replicates 123 Assign quantities to Standard wells 126 7 Setting Up the Thermal Profile 127 Set up the thermal profile 128 Elements of a Thermal Profile 129 Edit the thermal profile 131 Export the thermal profile image 140 8 Running and Monitoring Experiments 141 Overview of the Instrument Explorer dialog box 142 Add instruments to your network 143 Add a new instrument based on its IP address 143 Add a new instrument based on its port number 144 View information about an instrument 144 Start stop orpause arun 146 Startarun 146 Cancelarun 147 Pausearun 148 Monitorarun 149 Connect to the running instrument 149 Monitor a run by viewing its progress through the thermal profile Monitor a run by viewing the raw data plots 150 Agilent AriaMx Real Time PCR Program 150 Change the display options for the raw data plots Stop monitoringarun 153 Retrieve run data from the instrument 154 Retrieve data through the network 154 Retrieve data using a USB drive 155 Export instrument data to a CSV file 156 Export instrument data by column 156 Export instrument data by target 156 Export instrument data by wells 156 9 Setting Analysis Criteria 157 Ov
254. riaMx Real Time PCR System Setup and User s Guide If you are running the AriaMx ET software component you have access to the following electronic tracking functions e Controlled access to the AriaMx software through identification of all application administrators and users each with a unique username and encrypted password e Controlled database access through identification of all databases using a unique user ID and encrypted password e Experiment storage retrieval and deletion to from defined database s e Chronological and permanent audit trail recording of administrator and user actions that create modify or delete experiments as well as error recording e Database management including adding removing and switching databases switching databases is available at login e Controlled access to a defined database through administrator and user management e Report generation for audit trail user account and error logs e Report generation for experiment results with electronic signature e signature The help topics listed below provide information and instructions on using the program features that are unique to the ET component Open an experiment in the AriaMx ET software on page 286 Import and export experiments in the AriaMx ET software on page 289 Lock or log out of the AriaMx ET software on page 291 Change your password in the AriaMx ET software on page 293 Create a multiple expe
255. riment Analysis Reports and Exporting Results 14 The dialog box closes and the preview on the Export Data screen includes your changes Export to Excel When you export data results to Excel the program automatically launches Microsoft Excel and creates a workbook with each data item displayed on a separate tab within the workbook You can then save the workbook with the file name and folder location of your choice To configure and export data to an Excel file 1 On the Export Data screen next to File Type select Excel 2 Under Items mark the items that you want to include in the file See Select the items to include in the file on page 272 A preview of the file appears in the center of the screen 3 Click Export Data Microsoft Excel opens to the new workbook 4 In Excel save the file as desired Export to text files When you export data results as a text file the program creates a separate text file for each data item included in the data export configuration The file names include the project name and data item The program prompts you to select a folder location for the files You can then open the files in the text editing program of your choice To configure and export data to text files 1 On the Export Data screen next to File Type select Text 2 Under Items mark the items that you want to include in the file See Select the items to include in the file on page 272 3 Click Export Data The Brow
256. riment analysis project in the AriaMx ET software on page 294 Agilent AriaMx Real Time PCR Program Help for the AriaMx ET Electronic Tracking Software 16 Overview of the AriaMx ET software For administrators the following help topics provide information on administrative functions Manage users in the AriaMx ET software on page 295 Archive and restore experiments in the AriaMx ET software on page 302 View audit trails and system logs in the AriaMx ET software on page 306 Add and remove databases in the AriaMx ET software on page 311 View transaction logs in the AriaMx ET software on page 315 Agilent AriaMx Real Time PCR Program 285 16 Help for the AriaMx ET Electronic Tracking Software Open an experiment in the AriaMx ET software The AriaMx ET program tracks the actions taken on a post run experiment using a primary database with controlled access If the experiment you want to open is stored in the primary database you can open it directly from the database If you want to open a post run experiment that is saved to a file system i e a local or network folder you first must import the experiment into the database The instructions in this help topic describe how to open experiments from the primary database See Import and export experiments in the AriaMx ET software on page 289 for instructions on importing experiments Open an experiment You can open an experiment using mul
257. rmalizer and target of interest when calculating the relative quantities This method is a good choice for Comparative Quantitation experiments in which the normalizer and target of interest differ in amplification efficiency The Pfaffl method does not however incorporate run to run variations in amplification efficiency For a reference see Pfaffl M W 2001 Nucleic Acids Res 29 9 e45 This method is only available if the wells selected on the Analysis Criteria screen include a normalizer target Enter the amplification efficiencies for the targets If you selected the Pfaffl algorithm method to calculate the relative quantities you need to enter the amplification efficiency of each target If you determined the efficiencies based on a standard curve that was run in another experiment you can enter the efficiencies manually If the current experiment includes a standard curve to determine the efficiencies you can specify to use the efficiencies derived from that data Agilent AriaMx Real Time PCR Program Viewing Graphical Displays of the Results 10 To enter the amplification efficiencies manually 1 Select the Relative Quantity chart on the Graphical Displays screen 2 Inthe right panel click the downward arrowhead above the result table to display the advanced analysis parameter settings 3 Next to Mode select Pfaffl to enable the fields in the Amplification Efficiencies table 4 Inthe Amplification Efficiencies table en
258. s In the table at the top of the dialog box select an experiment or snapshot see Open and navigate the audit trail logs above for detailed instructions Click Print The Print dialog box opens Select a printer and click Print The program prints a copy of the table Agilent AriaMx Real Time PCR Program Help for the AriaMx ET Electronic Tracking Software 16 To export the table to Excel 1 Open the Log Viewer dialog box to the Audit Trail Logs 2 Inthe table at the top of the dialog box select an experiment or snapshot see Open and navigate the audit trail logs above for detailed instructions 3 Click Export to Excel Excel launches with a copy of the table displayed 4 Save the Excel file if desired View the system logs The system logs show the actions taken to manage user account information as well as any failed login attempts For each action item the logs indicate the user who performed the action the date and time of the action and a description of the action The system logs also show the total number of experiments that have been archived or restored and any failed attempts to archive or restore an experiment For failed archive or restore attempts the logs list the experiment name and the failure error message A Log Viewer L a 2 Log Audit Trail Logs System Logs Time Stamp User Description 24 Mar 2014 03 27 54 Admin Password of user Admin changed 24 Mar 2014 11 24 32 Adm
259. s See the topics below for instructions specific to each graph View the Amplification Plots on page 173 View the Melt Curve Raw Derivative Curve on page 183 View the Melt Curve Difference Plots on page 189 View the Standard Curve on page 196 View the Relative Quantity on page 201 View the Allele Determination graph on page 206 Agilent AriaMx Real Time PCR Program Creating Opening an Experiment 4 7 Export the results e To generate a report of the results navigate to the Generate Report screen click Generate Report in the Experiment Area panel on the left side of the screen Configure and create the report according to your selections e o export numerical data from the experiment navigate to the Export Data screen click Export Data in the Experiment Area panel on the left side of the screen Select the file type and information you want to export Agilent AriaMx Real Time PCR Program 45 4 Creating Opening an Experiment Create a new experiment Ab The Getting Started screen allows you to create a new experiment If creating the new experiment from scratch you start by selecting the experiment type If creating the new experiment from a template you start by selecting the template you want to use Once created you can set up the plate and thermal profile for the new experiment To open the Getting Started screen At the top of the program window click File gt New or
260. s displayed in the panel on the right side of the screen Agilent AriaMx Real Time PCR Program Viewing Graphical Displays of the Results 10 Adjust the graph properties You can adjust certain properties of the Standard Curve graph e g the scales of the axes the graph title and the background color through the short cut menu and the Graph Properties dialog box See Customize graph properties on page 211 for more information Manually adjust threshold fluorescence values The threshold fluorescence value for a target determines the target s Cq value The Cq is the cycle number at which the fluorescence level passes the threshold You can manually adjust the threshold fluorescence values while viewing the standard curves To adjust the threshold fluorescence value for a target by typing the desired value 1 Select the Amplification Plots graph on the Graphical Displays screen and unlock the threshold fluorescence values that you want to manually adjust 2 Select the Standard Curve graph 3 In the right panel click the downward arrowhead above the result table to display the advanced analysis parameter settings 4 Under Threshold Fluorescence type a new value into the field next to the desired target or click on the buttons to adjust the value The program adjusts the standard curves according to the new values To reset the threshold fluorescence value for a target back to the default value calculated by the pro
261. same value across all experiments in a project Review the three methods described below for setting threshold fluorescence levels than select the approach that best suits your experimental needs Method 1 Determine thresholds using a control reaction The most valid way to ensure equivalent thresholds between all the experiments in a project is to include a control reaction or reactions for each target on all the experiments in the project This reaction must have the same quantity of target in every experiment You can then manually adjust the thresholds in each experiment so that the well containing the control reaction has the exact same Cq in all experiments Use of this sort of inter experiment control is the most accurate way to set thresholds when performing multiple experiment analysis for a reference see Hellemans et al Genome Biology 2007 8 2 R19 If it is not possible to include a control reaction with each experiment follow one of the methods described below Method 2 Set the thresholds separately for each experiment This method is the default for setting the threshold levels in a project When you create a new project the program assigns a separate threshold using a background based algorithm to each target in each experiment based on the settings that you provide for the amplification plots Using this method the program bases the background based thresholds on noise levels in the baseline cycle range so this method
262. se For Folder dialog box opens 4 Select the folder where you want to save the text files and click OK The program creates the files and saves them in the designated folder Agilent AriaMx Real Time PCR Program 273 14 Generating Multiple Experiment Analysis Reports and Exporting Results 274 Load a saved data export definition If you already have a saved data export definition on your system that you want to use for the current project you can load that definition from the Export Results screen The saved definition must be for the same experiment type The program comes preloaded with a default data export definition that is automatically loaded You can also configure a custom definition for later use see Create or edit data export definitions below To load a data export definition e In the Export Configuration panel of the Export Data screen next to Definition select the saved data export definition from the drop down list The program updates the settings in the Export Configuration panel according to the selected definition After you load a definition you can still make edits to the data export configuration Create or edit data export definitions You can save your settings on the Export Data screen as a data export definition The program saves the definitions to your system allowing you to use them again with future projects of the same experiment type Save changes to the default data export defi
263. sed for amplification of the normalizer target 2 Inthe Normalizer Dye drop down list on the Properties panel select the dye that is to be used for detection of the normalizer target The program assigns the target name NORM to the selected wells and displays an N in the plate map 122 Agilent AriaMx Real Time PCR Program Setting Up the Plate 6 Assign Alleles Use the drop down lists under Allele to indicate which target is to be designated as Allele A and which target is Allele B To assign alleles 1 In the Dye Target Name drop down list for Allele A select the target that represents allele A 2 Inthe Dye Target Name drop down list for Allele B select the target that represents allele B Assign replicates Replicates are wells that contain identical reaction components repeats You can assign replicates using the Manual option or the Auto option When you designate replicate wells on the Plate Setup screen you can set the analysis criteria to average results from those wells or treat the wells separately When assigning replicates if you see a flashing red warning Icon in the Properties panel next to Replicates you have an invalid replicate set on the plate To be valid all the wells of a set must be of the same well type and have the same target assignments Hover your cursor over the warning icon to view specific information on why the program has called a replicate set invalid To assign replicates with the Auto
264. signed standard quantity from one or more wells e Select the well s and click Clear Agilent AriaMx Real Time PCR Program Setting Up the Thermal Profile Set up the thermal profile 128 Elements of a Thermal Profile 129 Edit the thermal profile 131 Export the thermal profile image 140 Agg Agilent Technologies 7 Setting Up the Thermal Profile Set up the thermal profile In the center of the Thermal Profile screen is a visual representation of the temperature cycling program that directs the instrument to incubate samples at specific temperatures for specific times To open the Thermal Profile screen With an experiment or project file open click Thermal Profile in the Experiment Area panel on the left side of the screen When you create a new experiment based on experiment type a default thermal profile is automatically assigned by the program The default thermal profile is one that is typical for the needs of that experiment type The exception is User Defined experiments Instead of assigning a default thermal profile the program prompts you to select a thermal profile from a set of defaults If you created the experiment from a template the default thermal profile is that of the template In any pre run experiment you can edit the thermal profile to fit your needs 128 Agilent AriaMx Real Time PCR Program Setting Up the Thermal Profile 7 Elements of a Thermal Profile Seqment titles Resolution 0 5 C Soak
265. sis parameter settings 3 Next to Normalization select On to turn on normalization or select Off to turn off normalization Adjust the range of the X axis You can quickly modify the range of temperatures displayed on the X axis To adjust the range of the X axis 1 Select the Melt Curve Raw Derivative Curve graph on the Graphical Displays screen 2 Inthe right panel click the downward arrowhead above the result table to display the advanced analysis parameter settings 3 Inthe fields below Temperature Range type in the desired lower temperature and upper temperature for the X axis range or click the buttons to adjust the temperatures in the fields to the desired values The program adjusts the range of the X axis accordingly Agilent AriaMx Real Time PCR Program Viewing Graphical Displays of the Results 10 Adjust product melting temperature settings Some of the settings that the program uses to identify amplification products and calculate melting temperatures are adjustable You can change the maximum number of amplification products for which the program reports a melting temperature When basing plots on the negative derivative of fluorescence you can set a minimum height that a product peak must reach in order for the program to consider it an amplification product To change the maximum number of amplification products for a which a melting temperature is reported 1 Select the Melt Curve Raw Derivative
266. solution 0 5 C SoakTime 10s The Ramp Mode dialog box opens Ramp Mode Settings Resolution C 0 2 Ey Soak Time s 10 Cancel 2 Next to Soak Time type in the desired number of seconds for the soak time or click the buttons to adjust the value in the field The soak time is the amount of time that the instrument spends at each temperature increment during the ramp 3 Click OK to close the dialog box and save your changes Restore the default thermal profile To restore the thermal profile to the default for the experiment type 1 Right click anywhere on the thermal profile display A short cut menu opens 2 Click Restore Default Thermal Profile The program sets the thermal profile to the default for the experiment type Agilent AriaMx Real Time PCR Program 137 7 Setting Up the Thermal Profile 100 80 60 Temperature C 40 20 138 Select segments for a blank thermal profile If you delete all segments in a thermal profile or if you create a new User Defined experiment the thermal profile screen appears as shown below Select segments to add in desired order Click Done when selections are complete or Start Over to clear selections and re select On this screen you select the segments you want to use to create a new thermal profile To select segments to add to a blank thermal profile 1 From the screen shown above click on the segment type that you want to be th
267. stinct after outlier wells are culled The percent distinct for the Population Distribution graph is displayed in the panel on the right side of the Graphical Displays screen next to Distinct Note that outlier wells are plotted on the Population Distribution graph with a red x Population Distribution 11000 10000 Plots for rows a 9000 A B C A Population ABC 5 8000 mli 7000 z ca 6000 s000 Plots for rows 4000 F G H Population FGH Columns If your qualification test failed Agilent s Technical Support staff may use the graphical data to help troubleshoot the cause of the failure 32 Agilent AriaMx Real Time PCR Program Performing Hardware and Software Tests and HRM Calibrations Perform an Installation Qualification test You can run an Installation Qualification IQ test to determine if the AriaMx software is properly installed on your system i wE ualifications Qualification Installation Operational Report C Users Public Documents Agilent AriaMx lQ_04 09 2014_ 11 43 22 pdf Change Tests Total Estimated Time 00 00 02 Name Requires Input GUI Software Installation Qualification Not Required Test Log Overall Result lt Not Available gt To perform the IQ test 1 Next to Report is the file path and file name of the report that the program generates at the end of the test To select a different folder or file name for the report click Change Then in the Save
268. stomize graph properties 211 Customize graph properties using the short cut menu 211 Customize graph properties using the Graph Properties dialog box 215 Agg Agilent Technologies 10 Viewing Graphical Displays of the Results Overview of the Graphical Displays screen 168 Graphs The Graphical Displays screen shows the results of the experiment displayed in a series of graphs For each graph the screen includes tools for setting certain analysis parameters The screen also includes a result table with configurable columns of data that you can export to an Excel spreadsheet To open the Graphical Displays screen Click Graphical Displays in the Experiment Area panel on the left side of the screen The Graphical Displays screen Is only available in post run experiments and in progress experiments that have completed at least one point of data collection The exact set of graphs available on the Graphical Displays screen varies depending on the experiment type but all experiments have a graph of the amplification plots and all experiments with a melt segment have a graph of the melt curves See the topics below for detailed information on specific graphs View the Amplification Plots on page 173 View the Melt Curve Raw Derivative Curve on page 183 View the Melt Curve Difference Plots on page 189 View the Standard Curve on page 196 View the Relative Quantity on page 201 View the Allele Det
269. t Agilent Technologies Click this link to open the About Agilent AriaMx message box This box displays the full version number of the software Contact Support Click this link to create a new email message directed to the Agilent Technical Support group Agilent AriaMx Real Time PCR Program 41 4 42 Creating Opening an Experiment About the ArialMx file types Three different files types can be created in the AriaMx program an experiment file a protocol template file and a project file These file types are summarized below Experiment Files amxd In the AriaMx program experiments of all types Quantitative PCR Comparative Quantitation Allele Discrimination and User Defined are saved as experiment files When an experiment file is open the program includes screens for defining the wells of the experimental plate setting up the thermal profile running the experiment and analyzing the results of that run Experiment files are given the extension amd Template Files amxt In addition to saving an experiment as an experiment file the plate setup and thermal profile of an experiment can be saved as a template file Creating a new experiment from a template file allows you to quickly set up new experiments that require a similar plate setup or thermal profile Template files are given the extension amt Project Files amxp Multiple experiment analysis projects are saved as project files Up to 8 pos
270. t run experiment files of the same experiment type can be added to a project enabling you to analyze the results side by side or combine results across experiments Project files are given the extension amxp Agilent AriaMx Real Time PCR Program Creating Opening an Experiment 4 Quick Start Protocol How to create set up run analyze and generate reports for an experiment 1 Create the experiment e Open anew tab On the Getting Started screen under New Experiment click Experiment Types to create a new experiment by the experiment type or My Templates to create a new experiment from a template 2 Set up the plate e After creating the experiment the experiment opens to the Plate Setup screen e Assign the plate properties based on experiment type including assigning well types replicate numbers and dyes targets See the topics below for instructions specific to each experiment type Assign plate properties for a Quantitative PCR DNA Binding Dye experiment on page 81 Assign plate properties for a Quantitative PCR Fluorescence Probe experiment on page 88 Assign plate properties for a Comparative Quantitation experiment on page 95 Assign plate properties for an Allele Discrimination DNA Binding Dye experiment on page 104 Assign plate properties for an Allele Discrimination Fluorescence Probe experiment on page 111 Assign plate properties for a User Defined experiment on page
271. t the data collection point for the amplification plots on the Analysis Criteria screen A lack of amplification plots would also occur if no data collection point was set in the amplification segment on the Thermal Profile Setup screen Without a data collection point no data is collected and analysis is impossible You will also not see any amplification plots if you selected ARn data when a reference dye was not defined for the experiment Signal fluctuations in amplification Possible sources of signal noise include environmental factors such as plots vibration of the bench from other instruments direct sunlight falling on the back of the instrument or fluctuations in the line power These problems can normally be resolved by relocating the instrument to a different bench in the lab or for the case of line power problems by connecting the instrument to a Power Conditioner or UPS Low increase in fluorescence with The probe is not binding the target efficiently Lower the annealing cycling temperature and verify the melting temperature Target PCR product is too long redesign primers to yield a PCR product lt 150 bp in length Magnesium concentration is too low run a titration to optimize concentration Insufficient or non specific product is being formed Verify product formation through gel electrophoresis Cq value reported for NTC Review amplification plot and adjust the threshold accordingly no target control sampl
272. t use two differentially labeled fluorescent probes to detect the two different alleles Each plotted point on the graph represents the coordinates of either the fluorescence values or Cq values for the two targets For example the X axis may correspond to the Cq of the Allele A target while the Y axis corresponds to the Cq of the Allele B target and the plotted point x y corresponds to the coordinates describing the two Cq values determined for a given well The position of the data point on the graph indicates the presence or absence of each allele To view the Allele Determination Click Graphical Displays in the Experiment Area panel on the left side of the screen If the Allele Determination graph is not already displayed click the icon for this graph at the bottom of the screen a g View data for a single data point If you set up the experiment to amplify the two alleles within the same well then each data point on the Allele Determination graph represents a single well or replicate set If you set up the experiment to amplify the two alleles in separate wells that contain the same sample then each data point represents a single sample To view a summary of the data for a single data point on a plot 1 Select the Allele Determination graph on the Graphical Displays screen 2 Hover your cursor over an individual data point on the graph A tooltip opens displaying the following information e The well ID or replicate number for
273. t which data collection points to analyze 249 Choose a treatment for replicate wells 250 Overview of the Graphical Displays screen for a project 251 Agilent AriaMx Real Time PCR Program 11 Graphs 251 Result table 252 Display options 252 Zooming 254 Compare amplification plots in a project 256 Compare amplification plots by experiment 256 Compare amplification plots by target 256 Consolidate the amplification plots 257 Compare raw or derivative melt curves in a project 258 Compare raw or derivative melt curves by experiment 258 Compare raw or derivative melt curves by target 259 Consolidate the raw or derivative melt curves 259 Compare standard curves in a project 260 Compare standard curves by experiment 260 Compare standard curves by target 261 Consolidate the standard curves 261 Compare Relative Quantity charts ina project 262 Compare relative quantities by experiment 262 Compare relative quantities by target 263 Consolidate the relative quantities 263 Compare Allele Determination graphs ina project 264 14 Generating Multiple Experiment Analysis Reports and Exporting Results Generate report of MEA project results 266 View a preview of the report 266 Select report type 266 Generate the report 266 Configure the report 267 Create or edit report configuration definitions 2 0 Export MEA data results to an Excel or text file 2 2 265 12 Agilent AriaMx Real Time PCR Program Configure the file and export data 2 2 Load a
274. tasks that require the use of the Instrument Explorer dialog box Add instruments to your network on page 143 Start stop or pause a run on page 146 Monitor a run on page 149 Retrieve run data from the instrument on page 154 Export instrument data to a CSV file on page 156 142 Agilent AriaMx Real Time PCR Program Running and Monitoring Experiments 8 Add instruments to your network The Instrument Explorer dialog box lists the instruments connected to your subnetwork If the instrument you need is not displayed in the list the dialog box has tools to search for and add a new instrument To open the Instrument Explorer dialog box At the top of the program window click Instrument gt Instrument Explorer Note that in order to start a run you must open the dialog box by clicking Run on the Thermal Profile Run Status or Raw Data Plots screen Starting a run is not enabled when you open the dialog box from the Instrument menu However opening the dialog box from the Instrument menu does allow you to monitor a run that has already been started Add a new instrument based on its IP address If you do not see the instrument you are looking for listed on the Instrument Explorer dialog box you can search for and add an instrument by its IP address You can look up the IP address of an instrument through the touchscreen Near the bottom right corner of the Home screen press the Connections button sho
275. te Setup in the Experiment Area panel on the left side of the screen In comparative quantitation it is important for the amplification efficiencies of the target of interest and the normalizer target to be reproducible and ideally very similar If you do not know the amplification efficiency for all of your targets run a standard curve See Determining amplification efficiencies for the targets of interest and normalizer targets on page 59 for more information Assign well types Use the Well Types drop down list to assign well types to all the wells used in the experiment See Well types for Comparative Quantitation experiments on page 61 for a description of the available well types To assign well types 1 On the Plate Setup screen select all the wells in the plate map that are of the same type For instructions on well selection see Select and view wells in the plate map on page 74 2 Select a well type from the Well Types drop down list in the Properties panel When the Show setting on the Properties panel is set to Type the well type appears at the top of the selected wells 3 Repeat steps 1 and 2 for all well types to be included in the experiment Agilent AriaMx Real Time PCR Program 95 6 96 Setting Up the Plate Assign well names After you assign wells to a well type you can if desired assign custom well names To assign well names 1 On the Properties panel of the Plate Setup sc
276. te Setup screen select a set of wells that are part of the same replicate set Make sure that the selected wells are of the same well type and contain identical targets For instructions on well selection see Select and view wells in the plate map on page 74 In the Properties panel under Replicates select Manual if not already selected Replicates CHM Auto Assign Replicate AA Number 6 Auto Increment ny In the Assign Replicate Number field type in the desired replicate number for the selected wells or click the buttons to enter the desired number The assigned replicate number appears in each selected well To assign replicates using Auto Increment 1 On the Plate Setup screen assign well types as needed for your experiment In the Properties panel under Replicates select Manual if not already selected Click Auto Increment When you hover your cursor anywhere on the plate map an icon of the number 1 appears next to the cursor 9 With the cursor click and drag across the group of wells that you want to assign as replicate number 1 Agilent AriaMx Real Time PCR Program Setting Up the Plate 6 The program assigns the wells to replicate number 1 and the icon next to the cursor changes to a number 2 5 Click and drag across the group of wells that you want to assign as replicate number 2 The program assigns the wells to replicate number 2 and the icon next to the cursor changes t
277. te number appears in each selected well Agilent AriaMx Real Time PCR Program 91 92 Setting Up the Plate 5 If desired make adjustment to the auto assignments in any of the wells of the plate by switching to the Manual option and manually assigning replicate numbers to those wells To manually assign replicates 1 On the Plate Setup screen select a set of wells that are part of the same replicate set Make sure that the selected wells are of the same well type and contain identical targets For instructions on well selection see Select and view wells in the plate map on page 74 In the Properties panel under Replicates select Manual if not already selected Replicates CHEI Auto Assign Replicate n Number B Auto Increment Maii In the Assign Replicate Number field type in the desired replicate number for the selected wells or click the buttons to enter the desired number The assigned replicate number appears in each selected well To assign replicates using Auto Increment 1 On the Plate Setup screen assign well types as needed for your experiment In the Properties panel under Replicates select Manual if not already selected Click Auto Increment When you hover your cursor anywhere on the plate map an icon of the number 1 appears next to the cursor 9 With the cursor click and drag across the group of wells that you want to assign as replicate number 1 Agilent AriaMx
278. ter the efficiency of each target using either of the following approaches e In the Slope column type the slope of the target or click the buttons to adjust the value in the field The program automatically adjusts the value in the Efficiency column by relating slope to amplification efficiency e In the Efficiency column type the amplification efficiency of the target or click the buttons to adjust the value in the field The program automatically adjusts the value in the Slope column by relating amplification efficiency to slope To enter the amplification efficiencies from a standard curve included in the experiment 1 Select the Relative Quantity chart on the Graphical Displays screen 2 Inthe right panel click the downward arrowhead above the result table to display the advanced analysis parameter settings 3 Next to Mode select Pfaffl to enable the fields in the Amplification Efficiencies table 4 Below the Amplification Efficiencies table mark the check box labeled Apply Std Curve Efficiencies to CQ Results Agilent AriaMx Real Time PCR Program 205 10 Viewing Graphical Displays of the Results View the Allele Determination graph For Allele Discrimination experiments and User Defined experiments that include wells with allele designations the Graphical Displays screen includes an Allele Determination graph This graph is useful for viewing the genotype results in Allele Discrimination experiments tha
279. termine the exact quantity of a particular DNA target in the experimental template samples This experiment type uses a standard curve produced with samples of known template quantity to derive the initial quantity of the target in the experimental sample For more information see The Quantitative PCR Experiment Type on page 56 The program offers two sub types for the Quantitative PCR experiment type DNA Binding Dye Including Standard Melt and Fluorescence Probe These sub types differ by the type of chemistry used to detect the PCR products In the DNA Binding Dye Including Standard Melt sub type detection is based on signal from a double stranded DNA binding dye e g SYBR Green and the default thermal profile includes a melt curve In the Fluorescence Probe sub type a target specific probe e g a TaqMan probe is used for target detection Comparative Quantitation The Comparative Quantitation experiment type is best used for comparing levels of RNA or DNA across samples when you do not require information about the absolute amount of target Most common is the comparison of amounts of mRNA in treated versus untreated or normal versus diseased cells or tissues The program will ask you to identify which wells contain the control sample called the calibrator and which wells contain the associated experimental sample called the unknown For accurate results you need to normalize the data to the quantity of a target g
280. the AriaMx ET software 286 Import and export experiments in the AriaMx ET software 289 Import experiments into the database 289 Export experiments from the database 289 Lock or log out of the AriaMx ET software 291 Lock the program 291 Log out of the program 291 Change your password in the AriaMx ET software 293 Create a multiple experiment analysis project in the AriaMx ET software 294 Manage users in the AriaMx ET software 295 Set account properties for all users 295 Manage user accounts 298 Archive and restore experiments in the AriaMx ET software 302 Archive experiments 302 Restore experiments 304 View audit trails and system logs in the AriaMx ET software 306 View the audit trail logs 306 View the system logs 309 Add and remove databases in the AriaMx ET software 311 Add an AriaMx ET database 312 Remove an AriaMx ET database 313 View transaction logs in the AriaMx ET software 315 View transaction logs for primary database 315 View transaction logs for an archive database 316 fide Agilent Technologies 16 Help for the AriaMx ET Electronic Tracking Software Overview of the AriaMx ET software 284 Agilent offers the AriaMx ET Electronic Tracking software component an optional upgrade providing 21 CFR Part 11 compatibility for users requiring security features such as user authentication database data storage and audit trail records For instructions on installing the ET version of the AriaMx software see the A
281. the AriaMx calibration algorithms were tested and optimized using the Agilent HRM Calibration Plate Using your own HCP reagent mixture may impact results Run an HCP All HCP experiments must be set up and run directly from the instrument you cannot set up an HCP experiment from the AriaMx program on your PC To run an HCP experiment 1 2 On the instrument Home screen press the HRM Calibration icon On the subsequent screen press Open Default Experiment The default HCP experiment opens to the Plate Setup screen All wells are set to the Unknown well type and the SYBR dye is selected for target detection in all wells The EvaGreen dye used in the Agilent HCP kit is detectable with the FAM SYBR optic module Navigate to the Thermal Profile screen and press Run Experiment A message box opens on the touchscreen prompting you to save the experiment Press OK in the message box to save the experiment to the HCP folder Select a file name for the experiment and press Save The instrument starts running the experiment After the run a message box opens on the screen notifying you if the HCP passed or failed e If it passed copy the post run HCP experiment file to your PC see Retrieve run data from the instrument on page 154 for instructions You can then use the HCP to calibrate HRM data from an experiment See Assign an HRM calibration plate on page 163 for instructions e If it failed you cannot use the
282. the wells that will be used for amplification of the normalizer target 2 Inthe Normalizer Dye drop down list on the Properties panel select the dye or dyes that is to be used for detection of the normalizer target The program assigns the target name NORM to the selected wells and displays an N in the wells on the plate map Agilent AriaMx Real Time PCR Program 99 100 Setting Up the Plate Assign replicates Replicates are wells that contain identical reaction components repeats You can assign replicates using the Manual option or the Auto option When you designate replicate wells on the Plate Setup screen you can set the analysis criteria to average results from those wells or treat the wells separately When assigning replicates if you see a flashing red warning icon in the Properties panel next to Replicates you have an invalid replicate set on the plate To be valid all the wells of a set must be of the same well type and have the same target assignments Hover your cursor over the warning icon to view specific information on why the program has called a replicate set invalid To assign replicates with the Auto option 1 On the Plate Setup screen select all the wells on the plate map that have the same number of wells per replicate set In the Properties panel under Replicates select Auto In the Direction of Assignment drop down list specify how the replicate wells are arranged on the plate e Select
283. tion definition as a new definition The program comes preloaded with a default report configuration definition which is loaded by default for new experiments When the default definition is loaded you can still make edits to the report configuration but you cannot save the changes to the default definition Instead save the report configuration as a new definition To save changes to the default definition as a new definition 1 With the default definition loaded on the Generate Report screen make your desired changes to the report configuration 2 Inthe Report Configuration Panel next to Definition expand the drop down list and click Add New The Add New Definition dialog box opens Agilent AriaMx Real Time PCR Program Generating Reports and Exporting Results 11 3 Inthe Definition Name field type a name for the definition or use the name provided 4 Make sure the Use Current Settings check box is marked Click Add The dialog box closes and the program saves the current report configuration to the new definition Save changes to a custom report configuration definition If you loaded a saved definition other than the default definition and then made changes to the report configuration you can save the changes either by overriding the existing definition or by creating a new definition To save changes to a report configuration by overriding the existing definition 1 After loading the saved definition on the G
284. tion series of the standard template sample Agilent AriaMx Real Time PCR Program 93 6 Setting Up the Plate 4 Inthe drop down list labeled A factor of select the dilution factor used to generate the dilution series of the standard template For example if each standard quantity is separated by a factor of 10 select 10x Negative dilution factors are used to specify a decrease in quantity from the starting amount while positive dilution factors specify an increase from the starting amount 5 In the drop down list labeled Units for Plate select the units of the quantity entered in the Starting Amount field Note that all Standard wells on the plate must use the same units To clear the assigned standard quantity from one or more wells e Select the well s and click Clear 94 Agilent AriaMx Real Time PCR Program Setting Up the Plate 6 Assign plate properties for a Comparative Quantitation experiment The program analyzes comparative quantitation data based on how the targets and well types are assigned in the Plate Setup screen For a particular target of interest the program measures the quantity of the target in an Unknown well relative to the level of that target in the Calibrator well To open the Plate Setup screen When you create a new Comparative Quantitation experiment you will automatically be directed to the Plate Setup screen To return to the Plate Setup screen at any time before during or after a run click Pla
285. tions are AR baseline corrected raw fluorescence ARn baseline corrected normalized fluorescence Set the Y axis scale for the Relative Quantity chart By default the program displays the Y axis in linear scale In this scale you can view the quantity of an target as a fold change in expression level relative to the calibrator This view is convenient for assessing increases in expression levels of a target relative to the calibrator Alternatively you can set the Y axis to display the target quantities on a base 2 logarithmic scale This view is convenient for viewing target expression levels that decrease or increase relative to the calibrator To display the relative quantities of the targets in the Unknown wells on a base 2 logarithmic scale 1 Select the Relative Quantity chart on the Graphical Displays screen 2 Inthe right panel next to Chart Type select Log2 The program updates the Y axis values on the chart To reset the chart to display relative quantities as a fold change relative to the quantities in the Calibrator wells 1 Select the Relative Quantity chart on the Graphical Displays screen 2 Inthe right panel next to Chart Type select Fold The program updates the Y axis values on the chart Agilent AriaMx Real Time PCR Program Viewing Graphical Displays of the Results 10 Add error bars to the Relative Quantity chart When you are treating replicate wells collectively you can select to display error
286. tiple approaches When you open an experiment you have the option to open the latest snapshot of the experiment i e the most recent version or an earlier snapshot of the experiment Refer to the image below for an explanation of how the program organizes experiments and snapshots in its browsers Experiment Name Date Username Admin Experiment name Quantitative PCR 10 Fold Example 2 items Snapshots of the Quantitative PCR 10 Fold Example 27 Mar 2014 07 15 09 Quantitative PCR 10 Fold Example 27 Mar 2014 07 16 46 experiment 286 Open the latest snapshot of an experiment You can open the most recent version of an experiment using multiple approaches To open the latest snapshot of an experiment from the Getting Started screen 1 Click the amp icon to the right of the tabs to open a new tab The new tab opens to the Getting Started screen 2 Click one of the options under Saved Agilent AriaMx Real Time PCR Program Help for the AriaMx ET Electronic Tracking Software 16 e To open an existing experiment that you recently accessed click Recently Opened In the bottom panel of the screen under Database is a list of post run experiments that you have recently opened Double click the experiment you want to open The program opens the experiment to the Plate Setup screen e To browse to the folder of the experiment click Browse Database The Browse Database dialog box opens with
287. tive Quantitation experiment you may designate two or more samples as biological replicates while assigning the sample names Samples that are biological replicates are assigned the same sample name but have different biological replicate ID numbers During analysis the program treats biological replicates independently as different samples If the experiment includes multiple biological replicates of the calibrator sample you can designate only one of the samples within the set of replicates as the calibrator You can change the assignment of the calibrator after the run if you want to re analyze the results using a different calibrator designation Agilent AriaMx Real Time PCR Program Selecting an Experiment Type 5 Well types for Comparative Quantitation experiments Well Type Description Unknown Contains a complete reaction mixture including a test template that contains an unknown amount of the target of interest Calibrator Contains a complete reaction mixture including an unknown amount of the target of interest The level of a target of interest in the calibrator wells is set to 1 0 for comparison to the relative quantities in unknown samples NTC No template control contains all reaction components except the template nucleic acid Standard Contains a complete reaction mixture including a known concentration of the target of interest This well type is used to generate a standard curve which is then used to relate the q
288. to capture the wells that you want to view in more detail 2 Minimap Agilent AriaMx Real Time PCR Program 77 6 Setting Up the Plate 4 Click the X in the upper right corner of the Minimap box to close the box The plate map on the Plate Setup screen displays only the wells selected in the Minimap box Standard G 1000 REF Standard Standard Standard Standard Standard G ieis REF Standard Standard Standard G ie 5 E REF Standard ie 17 REF Standard Standard Standard Standard Standard St andard 1 42 REF To restore the mini plate map to the full 96 wells 1 In the Properties panel click the Minimap icon The Minimap box opens 78 Agilent AriaMx Real Time PCR Program Standard Standard Standard e REF Standard Standard Standard iet REF Setting Up the Plate 2 Click the Maximize icon in the lower left corner of the Minimap box el 3 Click the X in the upper right corner of the Minimap box to close the box The plate map displays all 96 wells Agilent AriaMx Real Time PCR Program 79 6 Setting Up the Plate Export the plate map image The image of the Plate Setup screen s plate map can be exported to a Microsoft PowerPoint presentation To open the Plate Setup screen With an experiment or project file open click Plate Setup in the Experiment Area panel on the left side of the screen To export an imag
289. ty to each Standard well Standard Quantities Select Target es Starting Amount 0 000e 0 A factor of ix Units for Plate nanograms To assign the standard quantities for a target in the Standard wells 1 On the Plate Setup screen select the Standard wells For instructions on well selection see Select and view wells in the plate map on page 74 2 Inthe Properties panel under Standard Quantities select which target in the selected wells is the standard target i e the target of known quantity in the template 3 In the Starting Amount field type in the quantity of the standard target present in the first replicate set of Standard wells You will be asked to specify the units for this amount step 5 This quantity must be either the highest quantity or the lowest quantity in the dilution series of the standard template sample 4 In the drop down list labeled A factor of select the dilution factor used to generate the dilution series of the standard template For example if each standard quantity is separated by a factor of 10 select 10x Negative dilution factors are used to specify a decrease in quantity from the starting amount while positive dilution factors specify an increase from the starting amount 5 In the drop down list labeled Units for Plate select the units of the quantity entered in the Starting Amount field Note that all Standard wells on the plate must use the same units To clear the as
290. type 222 Generate the report 222 Configure the report 223 Create or edit report configuration definitions 226 Export data results to an Excel text or RDML file 228 Configure the file and export data 228 Load a saved data export definition 230 Create or edit data export definitions 231 215 10 Agilent AriaMx Real Time PCR Program 12 Creating and Setting Up an MEA Project 233 Quick Start Protocol 234 How to create set up analyze and generate reports for a multiple experiment analysis project 234 Overview of multiple experiment analysis 235 Applications 235 Restrictions 236 Guidelines for Comparing Cq Values Across Experiments 23 7 Reducing plate to plate variability 237 Selecting a method for setting threshold fluorescence levels 238 Create an MEA project 240 Open an existing MEA project 241 Select experiments fora project 242 Add or remove experiments from the project 242 Include or exclude experiments in the project analysis 243 Edit the plate setup of experiments ina project 244 Select an experiment to edit 244 Differentiate between targets across experiments 244 Edit plate properties 245 View the thermal profiles of experiments ina project 246 13 Analyzing Multiple Experiment Analysis Project Results 247 Set analysis criteria fora project 248 Toggle display between one experiment and all experiments 248 Select the wells and well types to include in analysis 248 Select the targets to include in analysis 249 Selec
291. uantification cycle Cq to initial template quantity in Unknown wells and calculate the amplification efficiency No reverse transcriptase control contains all QRT PCR reaction components except reverse transcriptase In one step RT PCR assays this control is useful for assessing levels of genomic DNA carryover that may contribute to fluorescence increase in the sample NAC No amplification control contains all reaction components except DNA polymerase Buffer Contains only buffer used to monitor the background fluorescence attributable to the buffer Agilent AriaMx Real Time PCR Program 61 5 Selecting an Experiment Type The Allele Discrimination DNA Binding Dye Experiment Type 62 The Allele Discrimination DNA Binding Dye experiment type is used to discriminate between two alleles of a gene in a genomic DNA or cDNA sample using a double stranded DNA binding dye such as SYBR Green or EvaGreen dye and a high resolution melt HRM HRM analysis is a technique used for genotyping samples that include a single nucleotide polymorphism SNP in the DNA sequence Applications that may use HRM analysis include species identification mutation screening and haplotype characterization When using the allele discrimination experiment type for HRM analysis you set up the experiment to amplify all alleles in the same well using the same set of primers and the program detects all alleles using the same double stranded DNA bind
292. ude a high resolution melt analysis to discriminate between the two product populations User Defined The User Defined experiment type provides the greatest flexibility in setup and analysis of an experiment All the well types and other plate setup options that are available across the other experiment types are available on the Plate Setup screen in a User Defined experiment Similarly on the Thermal Profile screen you can add any type of segment to the thermal profile and on the Graphical Displays screen you can view the results for any of the experiment type specific graphs Agilent AriaMx Real Time PCR Program 55 5 Selecting an Experiment Type The Quantitative PCR Experiment Type 56 In Quantitative PCR experiments the instrument detects the fluorescence of one or more dyes or fluorophores during each cycle of the thermal cycling process and a fluorescence value is reported for each dye fluorophore at each cycle Generally you want the instrument to acquire fluorescence readings during the annealing stage of thermal cycling You can quantify the initial copy numbers of RNA or DNA targets based on quantification cycle Cq determinations The Cq is defined as the cycle at which a statistically significant increase in fluorescence is first detected The threshold cycle is inversely proportional to the log of the initial copy number In other words the more template that is present initially the fewer the number of cycles
293. unknown sample using a standard curve from a separate experiment In a project that includes an experiment with a set of Standard wells the program can calculate the initial template quantity of a target in an Unknown well from a different experiment in the project This example walks you through the process of determining those initial template quantities Step 1 Create a project containing the experiments Create the project and make sure to add the two experiments to the project the experiment containing the unknown samples and the experiment containing the standard samples for the same target See Create an MEA project on page 240 for detailed instructions Step 2 Ensure target names are properly assigned The program can only compare the Unknown wells to the Standard wells if both well types have the same target name assigned to the same dye position The identical target name indicates that these wells amplified the same target View the target name assignments in each experiment and ensure that the target in the Unknown wells and the target in the Standard wells have been assigned the same name See Edit the plate setup of experiments in a project on page 244 Step 3 Select the analysis criteria Once you have ensured that the target name assignments are correct you can set the analysis criteria for the project on the Analysis Criteria screen Instructions on the controls and settings in this screen can be found in
294. ure including a test template that contains an unknown amount of the target of interest Buffer Contains only buffer used to monitor the background fluorescence attributable to the buffer NAC No amplification control contains all reaction components except DNA polymerase No template control contains all reaction components except the template nucleic acid This well type is useful for detecting amplicon contamination Standard Contains a complete reaction mixture including a known concentration of the target of interest This well type is used to generate a standard curve which is then used to relate the quantification cycle Cq to initial template quantity in Unknown wells and calculate the amplification efficiency No RT No reverse transcriptase control contains all QRT PCR reaction components except reverse transcriptase In one step RT PCR assays this control is useful for assessing levels of genomic DNA carryover that may contribute to fluorescence increase in the sample Agilent AriaMx Real Time PCR Program 57 5 Selecting an Experiment Type The Comparative Quantitation Experiment Type 58 The Comparative Quantitation experiment type provides an efficient method for comparing levels of RNA or DNA across samples when you do not require information about the absolute amount of target in any sample The most common application is the comparison of amounts of mRNA in treated versus untreated or normal versus dis
295. ustom well names shaw Type MEE Well Name 88 Agilent AriaMx Real Time PCR Program Setting Up the Plate 6 To assign well names 1 On the Properties panel of the Plate Setup screen next to Show select Name The Well Name field becomes available for typing 2 Select all the wells in the plate map that you want to assign to the same well name For instructions on well selection see Select and view wells in the plate map on page 74 You must assign a well to a well type before you can assign it a well name 3 In the Well Name field type the well name for the selected wells Press Enter The Well Name appears at the top of the selected wells 4 Repeat steps 2 3 for all well names that you want to assign Assign dyes targets Use the check boxes drop down lists and fields under Assign Dyes Targets to indicate which dyes are being used in each well and what target each dye is detecting Dye assignments are required but target name assignments are optional If different wells will be using the same dye to detect different targets assigning a unique name to each target enables the program to treat each target separately during analysis Add Dyes Targets 4 Use Dye Name Target Name O fa o C Ron O Hex O os O es O ber o Oc 0 0 Agilent AriaMx Real Time PCR Program 89 6 Setting Up the Plate To assign dyes and target names 1 On the Plate Setup screen select all the wells in the plate ma
296. vely These two options are part of the Grid Options sub menu Use these options to set the type of grid lines displayed on the graph When an option has a check mark next to it that indicates that it is turned on The absence of a check mark indicates that it is turned off When the Solid Grid Lines option is turned on the grid lines on the graph are solid lines When the Dashed Grid Lines option is turned on the grid lines on the graph are dashed lines This command which is part of the Grid Options sub menu opens the Graph Properties dialog box to the Grid Options tab You can customize the grid line color using the tools on this tab See Customize graph grid lines on page 217 This command opens the Graph Properties dialog box to the General tab You can customize the background color on the graph using the tools on this tab See Customize general graph properties on page 215 This command opens the Graph Properties dialog box to the General tab You can customize the graph title using the tools on this tab See Customize general graph properties on page 215 Agilent AriaMx Real Time PCR Program Viewing Graphical Displays of the Results 10 Print Image This command opens the Print dialog box where you can print a copy of the graph Save Image As This command opens the Save As dialog box where you can save a jpeg image of the graph Send Image to PowerPoint This command launches Microsoft PowerPoint and c
297. view wells in the plate map on page 74 In the Properties panel under Standard Quantities select which target in the selected wells is the standard target i e the target of known quantity in the template In the Starting Amount field type in the quantity of the standard target present in the first replicate set of Standard wells You will be asked to specify the units for this amount step 5 This quantity must be either the highest quantity or the lowest quantity in the dilution series of the standard template sample Agilent AriaMx Real Time PCR Program Setting Up the Plate 6 4 In the drop down list labeled A factor of select the dilution factor used to generate the dilution series of the standard template For example if each standard quantity is separated by a factor of 10 select 10x Negative dilution factors are used to specify a decrease in quantity from the starting amount while positive dilution factors specify an increase from the starting amount 5 In the drop down list labeled Units for Plate select the units of the quantity entered in the Starting Amount field Note that all Standard wells on the plate must use the same units To clear the assigned standard quantity from one or more wells e Select the well s and click Clear Agilent AriaMx Real Time PCR Program 103 6 Setting Up the Plate Assign plate properties for an Allele Discrimination DNA Binding Dye experiment The controls on Plate Setup scr
298. warning icon to view specific information on why the program has called a replicate set invalid Note that technical replicates are different from biological replicates The assignment of biological replicate IDs is unique to Comparative Quantitation and User Defined experiments Reference dye Passive reference dyes are used for normalization of the fluorescence signal in order to compensate for non PCR related variations in fluorescence such as pipetting variation from well to well Typically most experiments use ROX as the reference dye Agilent AriaMx Real Time PCR Program Setting Up the Plate 6 If you will be adding a dye to the wells of your plate as a passive reference dye you need to assign a reference dye in the plate setup The assignment of that dye as a reference dye is indicated in the wells by the target name REF Note that if you assign a reference dye you must assign it to all wells in use on the plate The Properties panel The panel on the right side of the Plate Setup screen has the tools for assigning properties to the wells in the plate map The content of this panel depends on the experiment type See the following help topics for information on your experiment type Assign plate properties for a Quantitative PCR DNA Binding Dye experiment on page 81 Assign plate properties for a Quantitative PCR Fluorescence Probe experiment on page 88 Assign plate properties for a Comparative Quantitati
299. well s and click Clear Agilent AriaMx Real Time PCR Program 6 87 6 Setting Up the Plate Assign plate properties for a Quantitative PCR Fluorescence Probe experiment The controls on Plate Setup screen s Properties panel allow you to create a customized plate setup for experiments of the type Quantitative PCR Fluorescence Probe To open the Plate Setup screen When you create a new Quantitative PCR experiment you will automatically be directed to the Plate Setup screen To return to the Plate Setup screen at any time before during or after a run click Plate Setup in the Experiment Area panel on the left side of the screen Assign well types Use the Well Types drop down list to assign well types to all the wells used in the experiment See Well types for Quantitative PCR experiments on page 57 for a description of the available well types To assign well types 1 On the Plate Setup screen select all the wells in the plate map that are of the same type For instructions on well selection see Select and view wells in the plate map on page 74 2 Select a well type from the Well Types drop down list in the Properties panel When the Show setting on the Properties panel is set to Type the well type appears at the top of the selected wells 3 Repeat steps 1 and 2 for all well types to be included in the experiment Assign well names After you assign wells to a well type you can if desired assign c
300. which axes of the graph have grid lines 1 Open the Graph Properties dialog box to the Grid Options tab 2 Next to Grid Lines select one of the options described below e Both Displays grid lines for the X and Y axes e X Axis Displays grid lines for the X axis only e Y Axis Displays grid lines for the Y axis only e None Does not display grid lines for either axis 3 Click Close The dialog box closes and the program adjusts the grid lines according to your Selection Agilent AriaMx Real Time PCR Program 217 10 Viewing Graphical Displays of the Results 218 To set the style of the grid lines 1 Open the Graph Properties dialog box to the Grid Options tab 2 Next to Styles select one of the options described below e Solid Displays the grid as solid lines e Dashed Displays the grid as dashed lines 3 Click Close The dialog box closes and the program adjusts the grid lines according to your selection To customize the color of the grid lines 1 Open the Graph Properties dialog box to the Grid Options tab 2 Expand the Color drop down list to view a menu of standard grid line colors 3 Select a color e If the standard menu includes your desired color click directly on it e If you need a color not included on the standard menu click Advanced to view an advanced menu for color selection Use the color picker tools to create a custom color The color menu closes and the new color is displayed in the Color drop dow
301. wn below The pop up box that opens includes the instrument s IP address z To search for and connect to an instrument based on its IP address 1 In the Instrument Explorer dialog box click the Add Instrument icon ob The Add Instrument dialog box opens 2 In the Instrument field type the IP address of the instrument you want to connect to then click Add The Add Instrument dialog box closes and the instrument is listed in the Instrument Explorer dialog box Agilent AriaMx Real Time PCR Program 143 8 Running and Monitoring Experiments Add a new instrument based on its port number If you do not see the instrument you are looking for listed on the Instrument Explorer dialog box you can search for and add instruments that are connected to the network on a different port number To select port numbers on which the application searches for available instruments 1 In the Instrument Explorer dialog box click the Discovery Port icon The Discovery Port dialog box opens The marked port numbers are the ones on which the program currently searches for instruments 2 Mark the port number you want to search e If the port number is already listed in the Discovery Port dialog box mark the check box in the Use column e If the port number is not listed click the Add icon to add additional ports e For any listed ports that you do not want included in the search clear the check box next to the port number or select the
302. ype For the Y axis of the amplification plots you can select to use fluorescence values that have been baseline corrected and or normalized to a reference dye if included To select the type of fluorescence data displayed in the amplification plots 1 Select the Amplification Plots graph on the Graphical Displays screen 2 Inthe right panel select one of the options next to Fluorescence Term The possible options are R raw fluorescence AR baseline corrected raw fluorescence Rn normalized fluorescence normalized to the reference dye ARn baseline corrected and normalized fluorescence If the thermal profile for your experiment included multiple data collection points during amplification see Select which data collection points to analyze on page 161 for instructions on specifying which data collection point to use for generating the amplification plots Specify the use of smoothing You can select to apply a curve smoothing algorithm to the amplification plots Smoothing alters the shapes of the amplification plots by decreasing the effects of signal noise The algorithm is based on a moving average calculation with 5 averaging points The smoothing option is turned on by default To turn smoothing on or off 1 Select the Amplification Plots graph on the Graphical Displays screen 2 Inthe right panel next to Smoothing select On to turn on smoothing or select Off to turn off smoothing 174 Agilent AriaMx
303. ype typically use a Standard curve to quantitate the amount of target present in an unknown sample with high accuracy using a fluorescently labeled probe or double stranded DNA binding dye for detection A series of Standard samples containing dilutions of a known amount of target are amplified to generate a curve that relates the initial quantity of the specific target to the Cq The standard curve is then used to derive the initial template quantity in Unknown wells based on their Cq values This method is sometimes referred to as absolute quantitation or as standard curve quantitation in the literature This experiment type is also useful for primer probe optimization experiments in the absence of a standard curve Comparative Quantitation Experiment Type This experiment type is a form of relative quantitation comparing the levels of a target gene in test samples referred to as Unknowns relative to a sample of reference referred to as the calibrator For example the calibrator sample might contain RNA from untreated cells while the Unknowns might contain RNA from cells treated with different experimental agents This experiment type provides an efficient method for comparing levels of RNA or DNA across samples when information about the absolute amounts of target in any sample is not required This method is used for establishing relative quantitation without the need for repeatedly performing a dilution series standard curve Allele Dis
304. ys in the Experiment Area panel on the left side of the screen See About the Qualification Test graphical data on page 31 for more information about the graphs on this screen 12 Check the panel on the right side of the screen to determine if the qualification test passed e If it passed the panel displays Results Pass e If it failed the panel displays Results Check If your qualification test failed contact Agilent Technical Support for help with troubleshooting See Contact Agilent Technical Support About the Qualification Test graphical data For a qualification test the Graphical Displays screen includes graphs for the Amplification Plots the Standard Curve and the Population Distribution The Population Distribution graph is unique to Qualification Test experiments For each row of Unknown wells on the plate this graph plots the number of initial template copies calculated for that row against the plate s column number Because all wells in rows A B and C are replicates and all wells in rows F G and H are replicates the distribution of the plot lines indicate the variability in initial template calculations across the thermal block within the two populations the ABC population Agilent AriaMx Real Time PCR Program 31 3 Performing Hardware and Software Tests and HRM Calibrations and the FGH population In order to pass the qualification test the program requires that both populations be at least 98 5 di

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