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Rotor-Gene® Q User Manual - Frank`s Hospital Workshop

1. 5 10 15 20 25 30 35 40 45 Cycle Options Page 1 v Normalise to Cycling A Yellow Normalise to Cycling A Orange Normalise to Cycling Red Crop start cycles Crop end cycles Adust Scale Auto Scale Detaut Scale Options This displays the drop down menu shown above which provides options tor transformation of the raw data Normalise to This enables normalization of amplification data to data from a passive reference dye such as ROX acquired in another channel 71 2 Rotor Gene Q User Manual 11 2012 Analysis User Interface Crop start cycles This creates a new channel data set in which some start cycles have been removed This is useful if large jumps are observed in the initial cycles which can occur when using certain chemistries Crop end cycles This creates a new channel data set in which some end cycles have been removed Page 1 This indicates the page that is currently selected to display the raw data plots The Edit Sample window allows the creation of multiple sample definitions For example data can be viewed with varying line thickness sample definitions and other display options This is particularly useful if relative quantitation is performed in a single channel because the user can easily switch the view between the gene of interest and housekeeper samples by defining 2 sample pages 7 4 Toggling samples At the right hand side of the main window is a toggl
2. Settings Given Conc Format 123 457 Unit r Samples ln gt Edit Reset Defaut Gradient En IH ame T ype G roups G iven Conc Optical Denaturation Ref Unknown 2 sample 1 untreated Unknown sample untreated Unknown 4 sample 1 untreated Unknown A5 Sample 1 treated Unknown AB Sample 1 treated Unknown A Sample 1 treated Unknown bet A8 Sample 2 untreated Unknown BE Sample 2 untreated Unknown 2 Sample 2 untreated Unknown Ex B3 sample 2 treated Unknown B sample 2 treated Unknown ES B5 sample 2 treated Unknown Sample 3 untreated Unknown An m eee Page Name Page 1 Ea a New Delete Synchronize pages OK Cancel This screen functions in the same way as the Edit Samples window Section 6 1 4 Normalization Normalization of EndPoint analysis data scales all signal levels to within the range of 0 1009 6 At least one positive and one negative control must be selected or more if analyzing multiple channels and the standards are not multiplexed More than one positive and one negative control should be run if there is a risk that a positive control may not amplify 1 For each channel all the positive controls are analyzed and the one with the highest fluorescence is set to be 10096 This means that if duplicate controls are run a positive control can fail without affecting the run 2 All the neg
3. Normalised Fluorescence Confidence Normalisation Regions mutation 100 00 Leading Range heterozygote 100 00 Start 82 4 mutation 95 72 End 828 heterozygote 97 58 heterozygote 37 80 Trailing Range wild type 100 00 Start 189 3 wild type 99 86 heterozygote 98 91 End 89 7 d heterozygote 96 23 wild type 99 23 pe Percentage mild hine ar sa so Rotor Gene Series Software 1 7 Build 94 VIRTUALMODE 4 Autocalling of samples is achieved by clicking Genotypes Enter the name of the genotype followed by the sample number which is used as a positive control to call unknown samples automatically iol mutation wild type heterozygote For more details on HRM analysis see Section 11 Run menu Start Run This option starts the defined temperature profile with the current gain settings Before the run starts the Profile Run Rotor Gene Q User Manual 11 2012 7 65 Analysis User Interface Confirmation window appears graphical representation of the temperature profile is displayed along with the gain settings for each channel 7 7 2 Pause Run This option enables a run to be paused and resumed Pausing and resuming can seriously affect the results of a run For this reason a marker in the data will indicate that the run was paused and the length ofthe pause A message is also placed in the messages tab of the Run Settings window see page 7 65
4. Crimson BBllnm 460 Source Detector Gain Create New Fl Orirn EB rm E dit 61 10 Edit Gain GEO 9 710hp 7 Remove 21 Reset Defaults Cancel Apply This is the name of the channel This specifies the excitation wavelength of the source LED This specifies the detection wavelength and filter type nm band pass hp high pass This specifies the gain for that particular channel This feature enables creation of new channels Clicking Create New opens a window that asks for a new name source and detection filter The filters can be chosen using the drop down menu next to each window Rotor Gene Q User Manual 11 2012 7 69 Analysis User Interface Channels Green yellow orange and red channels are standard configurations for 4 channel multiplex detection Tube Layout If using a 72 Well Rotor samples can be arranged to closely match the labeling on a 9 x 8 block By default the tube layout tab allows samples to be labeled sequentially i e 1 2 3 This means that samples are labeled consecutively in the order in which they are placed in the Rotor Gene Q Alternatively samples can be labeled 1A 1B 1C etc This option can be useful if samples were set up with a multichannel pipet Security The security tab displays information about the run signature The run signature is an irreversible key that is regenerated
5. Paste icon pastes cells that had been selected with the copy command into the currently selected position on the grid The Excel icon brings up a dialog box that prompts for a file name and folder in which to save the sample information After pressing Save the Excel file is opened automatically The Append Overwrite icon changes the editing of cells in the Edit Samples window If overwrite is selected existing data is overwritten when editing If append is selected new data is added to the end of existing data when editing Samples can be defined as one of several types listed in the following table 11 2012 7 75 Analysis User Interface Sample type Description None No sample in that position NTC No template control Negative Control Negative control Positive Control Positive control Unknown Unknown sample to be analyzed Standard Standard values are used to construct a standard curve to calculate unknown sample concentrations Calibrator RQ A calibrator is assigned a value of 1 and all other sample concentrations are calculated relative to this sample 7 76 Rotor Gene Q User Manual 11 2012 Analysis User Interface Page This function allows the user to have different sample definitions and also separate experiments in the same run This is useful for analysis of different products in different channels Use the arrow buttons to move between the sample pages Use the
6. This run was stopped as machine errors occurred that could not be recovered from Please contact your distributor if this occurs again attaching a support archive file Comments and suggestions This error occurs when the software has detected the chamber temperature has increased to above a safe level and hence activated a safe protection procedure Turn off the instrument immediately and contact your distributor or QIAGEN Technical Service This error occurs when the software has detected that the thermistor is open and therefore cannot read the temperature the software has then activated a safe protection procedure to turn off the machine Turn off the instrument immediately and contact your distributor or QIAGEN Technical Service This error occurs in the middle of the run after the software has made all possible attempts to recover and failed Further investigations are required by a QIAGEN Field Service Specialist to diagnose the problem with the instrument Please contact your distributor or QIAGEN Technical Service Rotor Gene Q User Manual 11 2012 12 7 Troubleshooting This page intentionally left blank 12 8 Rotor Gene Q User Manual 11 2012 Glossary 13 Term Acquisition Bins Channel Gain Gain Optimisation Loading Block Locking Ring Glossary Description Acquisition is the collection of fluorescent data Each acquisition set of fluorescent data from a channel is
7. To save disk space only Log Archives of the 60 most recent runs are stored Older run Log Archives will be overwritten as new run log archives are created 12 2 HRM troubleshooting Comments and suggestions Unable to run HRM Rotor Gene Q model is Contact your local QIAGEN representative not HRM equipped No HRM data obtained Incorrect setup Check filter settings Check if the rotor type is correct Check if the correct reagents have been used Check if the reaction was set up correctly Run a positive control experiment i e an assay that is known to provide results Rotor Gene Q User Manual 11 2012 12 1 Troubleshooting Comments and suggestions Plots look jagged Poor or no Check if the correct protocols and reagents have amplification been used We recommend QIAGEN kits for HRM analysis Check if the reaction was set up correctly Check the cycling conditions Check the starting quality and quantity of the template We recommend QIAGEN kits for sample preparation Amplification or melt plots are saturated Gain set too high Use Auto Gain Optimisation see page 6 24 Confidence percentages have changed Normalization regions Only move normalization regions if it is necessary were moved by clicking to avoid parts of the melt curve and dragging Outliers are present in the data Inconsistent reaction Check if the correct reagents have been used SUP Check that the tubes used are uniform Inhibit
8. Operating Procedures Software 6 1 2 Confirm profile The cycling conditions and acquisition channels of the template chosen are imported These can be altered using the Edit Profile window see Section 6 2 4 To initiate a run click the Start Run button It is also possible to save the template before starting the run by clicking on Save Template x 1 Rotor Selection 2 Confirm Profile amp New Open Save As Help The run will take approximately 77 minute s to complete The graph below represents the run to be performed Click on a cycle below to modify it Insert after Cycling Melt Insert before Remove Hold Temperature 95 deg Hold Time 2 mins 0 secs lt Back Save Template Start Run Cancel Rotor Gene Q User Manual 11 2012 6 5 Operating Procedures Software 6 1 3 Save run After clicking the Start Run button the Save As window appears The run can be saved in the user s desired location The run is given a file name that consists of the template used and the date of the run A serial number 1 2 etc is also included in the file name to allow automatic naming of numerous runs that use the same template on the same day Note The total path name should not exceed the operation system limit of 260 characters For example C program files rotor gene experiment files filename rex should not be longer than 260 character
9. closed tube system that prevents contamination with PCR products Results are comparable to conventional methods such as SSCP DHPLC RFLP and DNA sequencing 11 1 Instrumentation The Rotor Gene Q provides the following demanding real time and thermo optical capabilities required for HRM High intensity illumination Highly sensitive optical detection Fast data acquisition Finely controlled sample temperature Minimal sample to sample thermal and optical variation 11 2 Chemistry QIAGEN offers the Type it HRM PCR Kit for analysis of SNPs and mutations using HRM and the EpiTect HRM PCR Kit for methylation analysis Both kits contain the third generation intercalating fluorescent dye EvaGreen The kits combine optimized HRM buffer and HotStarTag Plus DNA Polymerase to avoid unspecific amplification products and provide reliable results Note All QIAGEN HRM kits and reagents are indicated for use with Rotor Gene Q instruments only for the applications described in the respective QIAGEN Kit handbooks 11 3 SNP genotyping example In the example shown the Type it HRM PCR Kit was used in HRM analysis to differentiate between homozygous wild type homozygous mutant and heterozygous forms of the human SNP rs60031 276 For technical details consult the Type it HRM PCR Handbook White H and Potts G 2006 Mutation scanning by high resolution melt analysis Evaluation of Rotor Gene 6000 Corbett Life Science HR 1 and
10. europ enne Plaque signal tique Label CSA pour le Canada et l arri re de l appareil les Etats Unis Plaque signal tique E Fabricant l gal l arri re de l appareil Plaque signal tique F Symbole DEEE pour l Europe l arri re de l appareil Label FCC de la Federal Communications Commission des Etats Unis Plaque signal tique l arri re de l appareil Label C Tick pour l Australie identifiant du fournisseur N17965 Label RoHS pour la Chine restriction de l utilisation de Plaque signal tique certaines substances l arri re de l appareil dangereuses dans l quipement lectrique et lectronique Plaque signal tique l arri re de l appareil B 12 Rotor Gene Q User Manual 11 2012 Appendix C Appendix C Safety Information German DE 1 Sicherheitshinweise Lesen Sie dieses Handbuch sorgf ltig durch bevor Sie das Rotor Gene Q benutzen Beachten Sie dabei insbesondere die Sicherheitshinweise Die Gebrauchsanweisungen und Sicherheitshinweise im Handbuch m ssen befolgt werden um einen sicheren Betrieb des Ger ts zu gew hrleisten und das Ger t in einem sicheren Zustand zu erhalten In diesem Handbuch werden die folgenden Kategorien von Sicherheitshinweisen verwendet WARNUNG Der Begriff WARNUNG WARNING weist Sie auf Situationen hin in denen eine Verletzungsgefahr f r Sie selbst oder andere Personen besteht N here Einzelheiten ber diese Situationen werden in einem T
11. 2 Cycling A Green 120 Page Page 1 i 2 Sample 2 3 Sample 3 P e e co e 77 5 Sample 5 5 Samples 0 7 gt Bank On Bank Off On AllOn All Off Edit Samples Fluorescence c 24 Temperature 60 zi Edit Gains Start Ia J By default all samples are shown in the display Samples can be removed from or added to the display using the toggler to the right The toggler consists of colored cells each of which corresponds to a sample in the display Samples with a brightly colored cell are displayed while samples with a faded cell are not displayed Samples can be switched on or off by clicking on the cell or by dragging the mouse pointer across several cells at time We recommend performing Manual Gain Adjustment as follows 1 Adjust the temperature in the Manual Gain Adjustment window to the acquisition temperature required for the run Rotor Gene Q User Manual 11 2012 6 29 Operating Procedures Software 6 30 Note The temperature will not be adjusted while the Rotor Gene Q is operating Restart the Rotor Gene Q to apply changes made to the temperature Click on Start The run begins The Rotor Gene Q temperature is adjusted to the temperature specified in the window The graphs in the window start to display data Wait for the temperature to stabil
12. 7 49 endpoint analysis 7 54 7 60 melt curve analysis 7 43 scatter graph analysis 7 51 Groups 7 81 H Hold 6 13 HRM advanced wizard 6 9 analysis 7 63 11 1 11 19 cycle 6 18 guidelines 11 7 kits 11 3 methylation analysis 11 5 primer dimers 11 18 quick start wizard 6 3 Rotor Gene Q User Manual 11 2012 Index 1 Index real time PCR 11 17 sample preparation 11 9 SNP genotyping 11 3 software 11 9 troubleshooting 12 1 Hybridisation 6 17 Ignore first 7 27 7 50 Informations de s curit B 1 Installation 4 1 grounding requirements 4 2 hardware 4 4 PC requirements 4 2 power requirements 4 2 site requirements 4 1 software 4 6 Intended use 2 3 L LinReg exporting to 7 8 Loading Block 5 4 Lock samples 7 99 templates 7 102 Locking Ring 36 Well Rotor 5 1 72 Well Rotor 5 2 Rotor Disc 100 5 3 Rotor Disc 72 5 2 Log archives 12 1 Long range 6 15 M Machine options 7 67 Maintenance 9 1 advanced wizard 6 9 Melt 6 17 Melt curve analysis 7 40 bins 7 42 peaks 7 42 Melt curve results window 7 43 Menu analysis 7 11 display options 7 83 file 7 5 gain 7 103 help 7 104 run 7 65 security 7 84 view 7 66 window 7 104 Messages 7 68 N Noise slope correction 7 26 7 50 Normalization 7 2 dynamic tube 7 26 7 50 endpoint analysis 7 57 Nucleic acid concentration measurement 6 3 7 61 O Operation conditions 1 5 1
13. 8 If you log in as administrator and remove all groups from the user CORIT198 linsongy the following message will appear when CORIT198 linsongy opens the software Rotor Gene Q Series Software You have insufficent rights to use the software Please contact the domain administrator to set up groups 7 9 2 Running multiple users on the same computer To use the Rotor Gene Q software with multiple users create a user account that does not have access to the Rotor Gene Q software Log into Windows using this account so that users cannot anonymously access the Rotor Gene Q 1 Using the Rotor Gene Software Login icon users can open their user account in the Rotor Gene Q software Rotor Gene Q User Manual 11 2012 7 95 Analysis User Interface t Rotor Gene Software Login 2 Enter the username and password in the box that appears Run Rotor Gene As Other User Domain CORIT188 LIF Username Cancel Password ns 3 The domain is either the computer you are logging into or the name of your local network Consult your network administrator if unsure which domain to enter in this field Note After logging in all of the user files will be available for that user Each user can save files in their own area This ensures a high level of security Note Each user should log out after their run has been completed to prevent other users from performing a run in
14. Acquiring Channels Crimson Green HAM Orange Red Yellow To acquire from a channel select it fram the list in the left and click gt To stop acquiring from channel select it in the right hand list and click lt To remove all acquisitions click lt lt Dye Chart gt gt Dye Channel Selection Chart FAM SYBR Green 17 Fluorescein Eva reen Alexa Fluor 4885 JOE VIC HEX TET CAL Fluor Gold 5402 Yakima Yellow ROX CAL Fluor Red 610 Cy3 50 Texas Red Alexa Fluor 568 i Dont Acquire Help 050 me M eue SEE Crimson 10hp uasar7 5 Alexa Fluor 680 SYTO 94 EvaGreen To set a channel to acquire select the channel and move it from the Available Channels list to the Acquiring Channels list using the gt button To remove a selected 6 16 Rotor Gene Q User Manual 11 2012 Operating Procedures Software channel from the Acquiring Channels list use the button The button removes all the channels from the Acquiring Channels list Clicking the Don t Acquire button also removes all acquisitions from the step If more than one cycling sequence is included in the profile the acquired data can be appended to the data acquired from the earlier cycling Use the Same as Previous drop down menu to select the cycling step to which the data should be appended The Dye Channel Selection Chart helps the use
15. formatted according to the currently selected location This drop down menu sets the units of measurement for the assay Samples gt Reset Detaut The style of the line can be moditied to improve readability of graphs on black and white printers Certain lines can be emphasized by moditying their style To access this feature click on the right arrow button next to the Edit button Pressing Edit opens the color selector Multiple rows can be selected when assigning a color to tubes Click Reset Default to reset all selected color cells back to their default color values Gradient allows a gradient to be chosen from the first to the last selected color Several gradients can be defined in a sample setup The New icon clears the Edit Samples window in preparation for data entry 7 74 Rotor Gene Q User Manual 11 2012 Analysis User Interface 8 icon s ICOn ICOn icon all Icon Icon Sample Types Rotor Gene Q User Manual The Open icon brings up a dialog box in which a Rotor Gene Q file can be selected to Import Note The number of samples in the open window and the file being imported must match The Save icon brings up a dialog box in which the name and folder can be entered in which a copy of the current sample definitions will be saved The Copy icon copies the selected cells The
16. 1 A 1 1 1 4 A 6 A 7 B 1 B B 1 B 2 B 4 B 6 Contents 5 Contents 1 4 S curit biologique 1 5 Produits chimiques 1 6 Mise au rebut des d chets 1 7 Dangers m caniques 1 8 Danger li la chaleur 1 9 Maintenance 1 10 Symboles du Rotor Gene Q Appendix C Safety Information German DE Sicherheitshinweise Sachgem e Handhabung Schutz vor Stromschlag Umgebungsbedingungen Biologische Sicherheit Chemikalien Entsorgen von Abf llen Gefahren durch mechanische Teile berhitzungsgefahr ON A 0 R WN Wartungsarbeiten 1 10 Symbole auf dem Rotor Gene Q Appendix D Quantitation Appendix E Rotor Gene Q products accessories and consumables Contents 6 Rotor Gene Q User Manual B 6 B 8 B 8 B 9 B 10 B 11 B 12 C 1 C C 1 C 2 C 4 C 6 C 6 C 8 C 8 C 9 C 10 C 11 C 12 D 1 D E 1 E 1 11 2012 Contents Appendix F F 1 Liability clause F 1 Index Index 1 Contents 7 Rotor Gene Q User Manual 11 2012 Contents This page intentionally left blank Contents 8 Rotor Gene Q User Manual 11 2012 Safety Information 1 Safety Information Before using the Rotor Gene Q it is essential that you read this user manual carefully and pay particular attention to the safety information The instructions and safety information in the user manual must be followed to ensure safe operation of the instrument and to maintain the in
17. 6 1 1 Rotor selection 6 4 6 1 2 Confirm profile 6 5 6 1 3 Save run 6 6 6 1 4 Sample setup 6 7 6 2 Advanced wizard 6 7 6 2 1 New Run Wizard window 1 6 9 6 2 2 Run Wizard window 2 6 10 6 2 3 New Run Wizard window 6 1 6 2 4 Edit Profile 6 12 6 2 5 New Run Wizard window 4 6 3 6 2 6 New Run Wizard window 5 6 31 Contents 2 Rotor Gene Q User Manual 11 2012 Contents 7 Analysis User Interface 7 1 7 1 Workspace 7 1 7 2 Toolbar 7 1 7 3 View raw channels 7 1 7 4 Toggling samples 3 7 5 File menu 5 7 5 1 New 7 5 7 5 2 Open and Save 7 7 7 5 3 Reports 7 9 7 5 4 Setup 7 9 7 6 Analysis menu 7 11 7 6 1 Analysis 7 11 7 6 2 Quantitation 7 12 7 6 3 Two standard curve 7 31 7 6 4 Delta delta C relative quantitation 36 7 6 5 Melt curve analysis 7 40 7 6 6 Comparative quantitation 7 44 7 6 7 Allelic discrimination 7 47 7 6 8 Scatter graph analysis 7 49 7 6 9 EndPoint analysis 7 52 7 6 10 Concentration analysis 7 61 7 6 11 High Resolution Melt analysis 7 64 7 7 Run menu 7 65 sd Start Run 7 65 7 7 2 Pause Run 7 66 7 7 3 Stop Run 7 66 7 8 View menu 7 66 7 8 1 Run Settings 7 66 7 8 2 Temperature Graph 7 71 7 8 3 Profile Progress 7 72 7 8 4 Edit Samples 7 73 7 8 5 Display Options 7 83 Rotor Gene Q User Manual 11 2012 Contents 3 Contents 7 9 7 9 1 7 9 2 1 7 3 7 9 4 9 5 7 9 6 7 10 7 11 7 12 Security menu Configuration Running multiple users on the same computer Audit trails Run Signatures S
18. Disc 100 Rotor and Locking Ring Rotor Disc 100 Loading Block Rotor Disc Pipetting Aid 30 individually wrapped discs for 3000 reactions 10 x 30 individually wrapped discs for 30 000 reactions For holding Rotor Disc 100 discs in the Rotor Gene Q requires Rotor Disc 100 Locking Ring For locking a Rotor Disc 100 in the Rotor Disc 100 Rotor Aluminum block for manual and automated reaction setup in Rotor Disc 100 discs Aid for marking well during manual reaction setup on a Rotor Disc Loading Block Heat sealing instrument for use with Rotor Discs requires Rotor Disc 72 or 100 Loading Block Rotor Gene Q User Manual Cat no 9241781 9242698 Inquire 981311 981313 9018895 9018896 9018909 9018897 9018898 11 2012 Product Rotor Disc Heat Sealing Film 60 Rotor Disc Heat Sealing Film 600 Rotor Disc 72 Starter Kit Rotor Disc 72 24 Rotor Disc 72 240 Rotor Disc 72 Rotor Rotor Disc 72 Locking Ring Rotor Disc 72 Loading Block Strip Tubes and Caps 0 1 ml 250 Strip Tubes and Caps 0 1 ml 2500 7 2 Well Rotor Locking Ring 72 Well Rotor Rotor Gene Q User Manual Contents 60 films for sealing Rotor Disc 100 or Rotor Disc 72 discs 10 x 60 films for sealing Rotor Disc 100 or Rotor Disc 72 discs Kit includes 3 Rotor Disc 72 packs Rotor Disc Heat Sealer Rotor Disc Heat Sealing Film Rotor Disc 72 Rotor and Locking Ring Rotor Disc 72 Loading Block
19. Edit Samples window page 7 70 Sample information may also be entered after the run has finished The Finish and Lock Samples button closes the screen and prevents the sample names from being modified For more information about this and other security features see Security menu Section 7 9 Rotor Gene Q User Manual 11 2012 6 31 Operating Procedures Software Hew Run Wizard Settings Given Conc Format Unit Copies More Options Samples E dit Reset Defaut Gradient EE im 2 Ir C ID Name Type Groups Given Cone Se a 1 JOE 3 Unknown Te 2 JOE E 36 Unknown Te 3 JOE E 30 Unknown Te 4 JOE E 3 NTC Unknown Te 5 Unknown Te Unknown Te Unknown Te Unknown Te Unknown m k Name Page 1 gt Mew Pelet Synchronize pages Skip wizard x Back Finish Finish and Lock Samples 6 32 Rotor Gene Q User Manual 11 2012 Analysis User Interface 7 1 7 2 7 3 Analysis User Interface This chapter describes the Rotor Gene Q software user interface Workspace The workspace is the backdrop of the main window In this area raw data plots and analysis results can be opened If several windows are opened simultaneously they can be organized by clicking the Arrange button on the toolbar There are several window arrangement options available that can be selected by clicking on the down arrow next to the Arrange button
20. Gene Q software provides sample locking Sample names can be locked by any user but can only be unlocked by an administrator For users who run their computers in administrator mode this option is of limited value To use this option the computer must be configured securely as described in the previous sections Note If you wish to lock samples do not run the software as an administrator Create an account with RG Operator and RG Analyst groups and keep the administrator password secret Users will then require authorization from the administrator to unlock files 7 100 Rotor Gene Q User Manual 11 2012 Analysis User Interface Samples can be locked before starting a run when using the Advanced wizard by clicking Finish and Lock Samples Hew Run Wizard Settings Given Conc Format Unit Copies Options Samples E dit Reset Defau Gradient ER G zs Ir C ID Name Type Groups Given Cone Se a 250bp Unknown Te 250bp Unknown Te 250bp Unknown Te E bp Unknown Te 006 Unknown Te E bp Unknown Te Unknown Te Unknown Te Unknown Re k Name Page 1 Mew Synchronize pages Skip wizard x Back Finish Finish and Lack Samples The following warning will appear Click Yes to confirm moo ID FE to Rotor Gene Q Series Software Ts Lock the samples Only administrators will be able to unlock them once this has been done Ga
21. New and Delete buttons to create and delete pages It is possible to have multiple sample definitions for the same channel in order to run multiple standard curves without multiplexing Simply define the samples of interest and their related standard curves on separate pages The single channel can then be analyzed with each set of definitions independently Sample pages can be labeled Page 1 Page 2 etc or they can be given any name e g Housekeeper This name will appear in reports When viewing the raw data the sample definitions used to display the data can be selected using the drop down menu next to the Options button 10 15 20 lt Scale Options Page 1 ar aE The sample page to use when performing an analysis can be selected in the Analysis window see Section 7 6 1 Rotor Gene Q User Manual 11 2012 7 77 Analysis User Interface Analysis 2 Std Curves Rel Other Quantitation kielt Cycling amp FAM Show Hide Auto shrink window Given Conc This shows the concentration for each of the standards The units can be defined as a decimal or log number If the standards are a dilution series it is only necessary to type the first 2 standards By pressing ENTER the program automatically adds the next logical dilution in the series Line style The style of the line can be modified to improve readability of graphs on black and white printers Certain
22. PCR product as the samples of interest and a detection chemistry for monitoring PCR product dissociation M An optical denature profile A new profile can be created or an existing profile can be edited see details below An Optical Denature Cycle appears almost identical to other cycles The principal differences are the melt step automatically inserted at the beginning of the profile and the sharp profile of the denature step during cycling The Optical Denature Cycle does not require defined hold times as the dissociation of the product is monitored at each cycle To perform this technique the following information about the run is required M The initial denaturation temperature This is the same temperature as the Denature step in a standard cycling profile M The tube position of the PCR sample that will produce a melt curve on the green channel M An Optical Denature Cycling profile must be defined Create a new Optical Denature Cycle as follows 1 Open the Edit Profile window Then click on New In the window that appears click the Insert after button 6 20 Rotor Gene Q User Manual 11 2012 Operating Procedures Software and select New Cycling from the menu Select one of the temperature steps by clicking on the graph In the drop down menu change from Timed Step to Optical Denature A default profile containing a Denature step and an Optical Denature Cycle step will appear The ramped
23. Q and use Gain Optimisation to determine the best gain setting If the gain chosen by Gain Optimisation results in a poor signal then the Target Sample Range should be increased If it results in a signal that is saturated then the Target Sample Range should be decreased To perform Gain Optimisation click on the Gain Optimisation button in New Run Wizard window 3 see Section 6 2 3 6 24 Rotor Gene Q User Manual 11 2012 Operating Procedures Software New Run Wizard Temperature Profile This box displays help on elements in the wizard Far help on an item hover your mouse over the item Far help ou ean also click on a combo box to display help about its available settings Edit Frofile Channel Setup Create New 4 l nm S10nm 53 nm 555nm als 1 Edit Gain B25nm 6b nm Lrmson Balinm 10 Remove 4 Onm 51 nm Gain Optimisation Skip wizard lt lt Back gt gt The Auto Gain Optimisation Setup window appears This window enables optimization by automatically adjusting the gain settings until the readings for all selected channels fall within or below a certain threshold Reset Defaults Rotor Gene Q User Manual 11 2012 6 25 Operating Procedures Software Auto Gain Optimisation Setup Optimization Auto Gain Optimization will read the Fluaresence on the inserted sample at AN differen
24. Q and click into place Close the Rotor Gene Q lid 10 2 Rotor Gene Q User Manual 11 2012 Optical Temperature Verification 3 Access the Advanced wizard by selecting the Advanced tab in the New Run window In the Advanced wizard click on Instrument maintenance and then OTV The wizard prompts for the OTV serial number This number can be read from the label on the OTV Rotor Disc or can be imported from the CD by clicking on Browse and choosing the otv file provided on the CD Once the number is entered click Start Rotor Gene Q User Manual 11 2012 10 3 Optical Temperature Verification Quick Start Advanced LE 5 Perform Last Run maintenance h Three Step with Melt om Two Step vl HRM W 3l Other Runs Instrument Maintenance Open A Template In Another Folder v Show This Screen When Software Opens F Optical Temperature Yerification Rotor se This template allows vou ta perform in tube temperature verification with the DIAGEM gt UTY Rotor For more information see our website available from the Help menu Rotor Details u Fluarescent insert placed corectly Please enter the serial number of the rotor you are using 0 TV L172 31FA 2D0 384 306 3B 30 Temperatures 50 72 30 Expiry Date 2006 Validity Check Start Exit 4 The software then prompts for a filename for the run Then the run begins 10 4 Rotor Ge
25. Rotor Disc Pipetting Aid 24 individually wrapped discs for 1728 reactions 10 x 24 individually wrapped discs for 17 280 reactions For holding Rotor Disc 72 discs in the Rotor Gene Q requires Rotor Disc 72 Locking Ring For locking a Rotor Disc 72 in the Rotor Disc 72 Rotor Aluminum block for manual and automated reaction setup in Rotor Disc 72 discs 250 strips of 4 tubes and caps for 1000 reactions 10 x 250 strips of 4 tubes and caps for 10 000 reactions For holding Strip Tubes and Caps O 1 ml requires Locking Ring 72 Well Rotor For locking Strip Tubes and Caps O 1 ml in the 72 Well Rotor 11 2012 Appendix E Cat no 981601 981604 Inquire 981301 981303 9018899 9018900 9018910 981103 981106 9018903 9018904 E 3 Appendix E Product Loading Block 72 x 0 1 ml Tubes Loading Block 72 x 0 1 ml Multi channel PCR Tubes 0 2 ml 1000 PCR Tubes 0 2 ml 10000 36 Well Rotor 36 Well Rotor Locking Ring Loading Block 96 x 0 2 ml Tubes Rotor Disc OTV Kit Rotor Holder E 4 Contents Aluminum block for manual reaction setup with a single channel pipet in 72 x 0 1 ml tubes Aluminum block for reaction setup with multichannel pipets in 72 x 0 1 ml tubes 1000 thin walled tubes for 1000 reactions 10 x 1000 thin walled tubes for 10 000 reactions For holding PCR Tubes 0 2 ml requires 36 Well Rotor Locking Ring For locking PCR Tubes 0 2 ml
26. Shaw All Analysis Options Show Hide Auto shrink window Auto shrink Selecting Auto shrink window shrinks the window window when it is not in use Moving the cursor over the window enlarges the window again Rotor Gene Q User Manual 11 2012 7 11 Analysis User Interface 7 6 2 Organizing the workspace Each time a new analysis is started its windows are arranged to fit in with those already on the screen If many windows are displayed this can be cumbersome Close the windows you do not require then click Arrange on the toolbar The windows are automatically arranged according to the Smart Tiling method Alternatively select another arrangement method by clicking the arrow next to the Arrange button Clicking the right mouse button on the name of an analysis also provides additional options Quantitation Melt x Cycling Green Page 1 Cycling Orange Show Cycling amp Red Hide Cycling Remove Analysis Show This displays the selected analysis Hide This hides the selected analysis Remove This removes the selected analysis Analysis completely This means that any normalization settings or melt bins set up in the analysis will be lost Quantitation Select the Quantitation tab in the Analysis window and then double click on the channel name or select the channel and then press the Show button to open the channel of inter
27. Type in your name and click Next The Internet E mail Address window will open Internet Connection Wizard 1 X Internet E mail Address Your e mail address is the address other people use to send e mail messages to you E mail address For example someone microsoft com lt Back Cancel Rotor Gene User Manual 11 2012 7 105 Analysis User Interface 2 Type in your E mail address and press Next The E mail Server Names window will open Internet Connection Wizard X E mail Server Hames My incoming mail server is a For3 Server Incoming mail POPS IMAP or server SMTP server i the server that iz used for your outgoing e mail Outgoing mail SMTP server lt Back Cancel 3 Select the type of mail server for incoming mails and specify the server names for incoming and outgoing e mails Then press Next The window Internet Mail Logon will open Internet Connection Wizard X Internet Mail Logon the account name and password your Internet service provider has given you Account name Password fr Remember password IF Your Internet service provider requires you to use Secure Password Authentication SPA to access your mail account select the Log On Using Secure Password Authentication SPA check Log on using Secure Password Authentication SPA lt Back Cancel 7 106
28. a Denature step This option may be chosen if a Gain Optimisation during the run would result in too much time spent on the initial step Usually Perform Optimisation Before 1st Acquisition is preferred because Gain Optimisation is performed as close as possible to the run conditions Channel This drop down menu allows channels to Settings be added Choose the channel of interest and click Add Edit This opens a window in which the Target Sample Range can be set The Target Sample Range is the range of initial fluorescence that should be set for the sample in the specified tube Auto Gain Optimisation reads each channel using gain settings in the range specified by Acceptable Gain Range It chooses the first gain setting that results in a fluorescence reading within the Target Sample Range In the example shown Auto Gain Optimisation searches for a gain setting between 10 and 10 that gives a reading between 5 and 10 FI in tube 1 In general tor intercalating dyes a Target Sample Range of 1 3 Fl is appropriate while a range of 5 10 FI is more suitable for probe chemistries Rotor Gene Q User Manual 11 2012 6 27 Operating Procedures Software Auto Gain Optimisation Channel Settings Channel Settings Channel Green Tube Position fi Target Sample Range 5 Fl up to 10 FI Acceptable Gain Range 10 to 10 eed Heo Remove Remove
29. a standard curve once validation has been performed 2 From the Other tab in the Analysis window select Delta Delta Relative Quantitation Select New Analysis 2 Std Curves Ael oe ee Allelic Discrimination Clearing Point Analysis Cycling amp Green swf Cycling amp Orange Pag Comparative Quantitation Concentration Analysis Delta Delta CT Relative Quantitation EndPoint Analysis High Resolution Melt Analysis Scatter Graph Analysis Show Al Analysis Options Show Hide Auto shrink window 3 Enter a name for the analysis Rotor Gene Q User Manual 11 2012 7 37 Analysis User Interface Rotor Gene Q Series Software E Enter a name for the relative quantitation analysis Cancel X _Cancel_ Relative Quant Analysi 4 Validation Run Performed must be checked to proceed with the analysis Define the pages where the gene of interest and normalizing gene have been analyzed Relative Quant Analysis Reports Export J Gene Expression H Delta Delta CT Relative Quantitation C Validation Run Performed Gene of Interest Quantitation Normaliser Quantitation Calibrator Defined 7 38 Rotor Gene Q User Manual 11 2012 Analysis User Interface fo Selection of Normaliser Standard Curve Currently Selected Hone To select an existing analysis to use or to create new analysis select a channel fram th
30. analysis ensure maximum specificity with no need for optimization Use sufficient preamplification template Analysis of real time PCR data can be very useful when troubleshooting HRM analyses Amplification plots should have a threshold cycle of less than or equal to 30 cycles Products that amplify later than this due to low starting template amount or template degradation typically produce variable HRM results due to PCR artifacts Normalize template concentration The amount of template added to the reaction should be consistent Normalize the starting concentrations so that all amplification plots are within C values of each other This ensures input concentrations are within a 10 fold range Rotor Gene Q User Manual 11 2012 11 7 High Resolution Melt Analysis Check for aberrant amplification plots Prior to running HRM examine amplification plot data carefully for abnormal amplification plot shape Plots with a log linear phase that is not steep is jagged or that reaches a low signal plateau compared to other reactions can indicate poor amplification or a fluorescence signal that is too low e g this could occur if primer concentration was too low Poor reactions can be caused by reaction inhibitors or incorrect reaction setup HRM data from such samples can be inconclusive or of low resolution To avoid unreliable results we recommend QIAGEN kits for sample preparation and HRM analysis Keep postampl
31. analyzer with 2 channels green yellow plus HRM channel laptop computer software accessories 1 year warranty on parts and labor Real time PCR cycler with 5 channels Inquire green yellow orange red crimson laptop computer software accessories 1 year warranty on parts and labor Real time PCR cycler and High Resolution Inquire Melt analyzer with 5 channels green yellow orange red crimson plus HRM channel laptop computer software accessories 1 warranty on parts and labor Real time PCR cycler with 6 channels Inquire blue green yellow orange red crimson including laptop computer software accessories 1 year warranty on parts and labor 1 year coverage of parts labor 9243310 shipping turnaround time 7 10 working days Rotor Gene Q User Manual 11 2012 E 1 Appendix E Product RGQ Full Agreement no PM RGQ Full Agreement Accessories Rotor Disc 100 Starter Kit Rotor Disc 100 30 Rotor Disc 100 300 Rotor Disc 100 Rotor Rotor Disc 100 Locking Ring Rotor Disc 100 Loading Block Rotor Disc Pipetting Aid Rotor Disc Heat Sealer E 2 Contents 1 year coverage of parts labor shipping availability of loaner system in 48 hours 1 year coverage of parts labor shipping availability of loaner system in 48 hours 1 on site inspection per year Kit includes 2 Rotor Disc 100 packs Rotor Disc Heat Sealer Rotor Disc Heat Sealing Film Rotor
32. at which the reference sample has denatured Tube Position Ramp fram 80 to 35 H deg Hold for rs mins secs Denature Offset fo Es deg c Cancel 4 Ensure that The tube indicated in Tube Position contains a PCR product that will show a melt peak on the green channel The final ramp temperature will not burn the sample yet will be high enough to allow it to melt m The hold time is sufficient to denature the sample m The denature offset is set appropriately The default of 0 C is appropriate for most melts Melts with very sharp transitions may require a denature offset of 0 5 C to 2 C as determined by the user to ensure that melt transition is detected You can also define a Denature step by introducing a new Hold step Click on Insert before and select New Hold at Temperature from the menu The calibration settings will appear 6 22 Rotor Gene Q User Manual 11 2012 Operating Procedures Software Hold Temperature 95 deg Hold Time 3 mins 0 secs Calibration Step M Calibration Settings Acquinng to RealTime an tube 1 Ramp fram 80 to 35 and hold for 3 mins secs Offset deg E dit The calibration settings are synchronized with the denature settings so a change to the hold time in the Denature step will automatically update the calibration hold time This is because the calibration process and denaturation are equivalent in Optical Denature
33. between these scales only alters the display of the graphs not the calculations This can be verified by 7 30 Rotor Gene Q User Manual 11 2012 Analysis User Interface 7 6 3 use of the pinpointer tool by right clicking on the graph and selecting Show pinpointer Using a log scale smaller values are more visible on the graph whereas a linear scale facilitates a view of the entire reaction Note Amplification plots update in real time as the Rotor Gene Q is actively acquiring data during a run This real time monitoring of data enables the user to see results as soon as the curves show exponential growth Preliminary conclusions can be drawn and decisions made for the next run Quantitation analysis templates Quantitation analysis templates allow the user to export normalization and threshold settings into a single qut file This file can be imported and reapplied in other experiments See Section 8 1 for more details Imported Settings lt none gt Import Export Two standard curve Relative gene expression analysis using a normalizing gene can be performed using the 2 standard curve method The method requires a standard curve for each gene The concentration for each gene is quantified according to its standard curve The expression of the gene of interest is then normalized with the normalizing gene often a housekeeping gene It is important that the standards and replicate samples are design
34. cycles may not be indicative of the background level just prior to amplification In contrast dynamic tube normalization uses the second derivative of each sample trace to determine a takeoff point for each sample The background level is then averaged from cycle 1 up to this takeoff cycle number for each sample This gives the most precise quantitation results Note that for some data sets background fluorescence is not consistent during the cycles before amplification commences In these cases it may be necessary to deselect dynamic tube normalization by clicking on Dynamic Tube because it could result in less precise quantitation Noise slope correction The background fluorescence Fl of a sample should ideally remain constant before amplification However sometimes the Fl shows a gradual increase or decrease over several cycles due to the chemistry used This produces a skewed noise level Noise slope correction uses a line of best fit to determine the noise level instead of an average and normalizes to that line Selecting this option by clicking the Slope Correct button can improve data from replicates if sample baselines are noticeably sloped Noise slope correction improves the data when raw data backgrounds are observed to slope upward or downward before the takeoff point 7 26 Rotor Gene Q User Manual 11 2012 Analysis User Interface Ignore First The fluorescence signal from the first few cycles in a ru
35. displayed in the software as unanalyzed data in a Raw channel window This data can be analyzed using the options in the Analysis menu In a melt analysis bins are set to define a region where a melt peak is expected to occur Genotypes can be defined based on the presence of peaks in certain bins or combinations of bins A channel consists of a light emitting diode LED with an excitation filter paired with an emission filter The LED and excitation filter excite samples at a given wavelength Fluorescence emitted by samples is passed through the emission filter before being detected by a photomultiplier The Rotor Gene Q uses a photomultiplier to collect fluorescence photons and convert them to electronic signals The gain is a setting that determines the sensitivity of the photomultiplier If the gain is set too high the signal is oversaturated If the gain is set too low it is not possible to differentiate signal from background noise Gain Optimisation is a process that dynamically adjusts the gain setting allowing an appropriate setting to be selected which results in optimal signal detection Loading Blocks are aluminum blocks available in different formats which are used to hold tubes or Rotor Discs during reaction setup Rotor Disc Loading Blocks are also used with the Rotor Disc Heat Sealer to heat seal Rotor Discs Locking Rings are metal rings that fit onto the rotor to prevent tubes and caps from coming loo
36. distributor Select the correct communication port to enable communications between the computer and the Rotor Gene Q If you are unsure which port to select click Auto Detect to search for all available ports If you are unsure which port to select click Auto Detect to search for all available ports 7 10 Rotor Gene Q User Manual 11 2012 Analysis User Interface 7 6 Analysis menu 7 6 1 Analysis After clicking Analysis the Analysis window appears This window allows creation of new analyses and display of existing analyses The method of analysis is selected using the tabs A list of the channels that can be analyzed using the selected method is shown Multiple assays run in the same channel can be analyzed independently provided they have been set up as separate pages in the Edit Samples window Pages that have already been analyzed have a green checkmark next to them This means that threshold and normalization settings have been saved for this analysis To view or analyze a channel double click on it The specific analysis window appears Analysis xl 2 Std Curves Rel Other Quantitation Melt Delta Delta CT Relative Quantitatian Allelic Dizcriminatian Cycling amp Green Comparative Quantitation Scatter Graph Analysis EndPoint Analysis Cycling Orange Cycling Cycling 4 Yellow Concentration Analysis High Resolution Melt Analysis
37. every time the file is changed If any section of the rex file is modified outside the software the signature and the file will no longer match Checking the signature allows confirmation that the raw data was not modified outside the application that the profile has not been tampered with and that the temperature graph is valid The signature also protects against corruption such as file system errors Note If rex files are e mailed the encryption process can invalidate the signature To avoid this zip the file before e mailing 7 70 Rotor Gene Q User Manual 11 2012 Analysis User Interface Run Settings Xx General Information Hun Signatures are stored within all newly saved runs These signatures like a wax seal on a document guarantee that no changes have been made outside the software If a file is tampered with the signature becomes invalid Hun Signature The signature is valid A The signature for this run file is walid The file contents has not been modified outside af the software Cancel Apply 7 8 2 Temperature Graph Select Temperature Graph from the View menu or click on the Temp button to bring up the Temperature window The graph displays the course of the set temperatures during cycling It does not reflect a real time temperature measurement As the run proceeds the Set Actual and Hold time is shown for each step of the program For an existing run
38. for the run Complete the fields clicking Nest when you are ready to move to the nest page Operator 3 pour mouse over the item for help ou Mates also click on a combo to display help about its available settings R action 2l Volume pL Sample Layout KEREN Skip wizard lt lt Back Next gt gt 6 2 3 New Run Wizard window 3 In this window the Temperature Profile and Channel Setup can be modified Ifthe Edit Profile button is clicked the Edit Profile window appears enabling alteration of cycling conditions and selection of acquisition channels Section 6 2 4 After setting up the profile click the Gain Optimisation button to bring up the Gain Optimisation window see page 6 24 Rotor Gene Q User Manual 11 2012 6 11 Operating Procedures Software Hew Run Wizard Temperature Profile the wizard Far help on an item hover Your mouse over the item Far help ou can also click on a combo bos to display help about its available settings Edit Profile Channel Setup Create New 4 linm 1l nm E Edit 53 nm 555nm 585mm 61 Orr Edit Gain 625rnm bb nm BB nm 710hp Remove Ainm SlUnm Reset Defaults Gain Optimisation Skip wizard x Back Next gt gt 6 2 4 Edit Profile The Edit Protile window allows the cycling conditions and acqu
39. genotype This is used when printing multichannel genotyping reports so that all results from multiple channels can be easily read For multiplex analysis genotypes must be set up in each channel If for example a dual channel quenched FRET analysis is run where a wild type and heterozygous genotype are expected in each channel the bin parameters must be set up for each channel The results will then be given in a multiplex report Melt analysis templates Melt analysis templates allow the user to export normalization threshold genotype and bin settings into a single met file This file can be imported and reapplied in other experiments See Section 8 1 for more details Imported Settings amp gt Import Export Comparative quantitation Comparative quantitation compares the relative expression of samples to a control sample in a run when a standard curve is not available This is frequently used in microarray analysis Warton and coworkers 2004 provide an example of this technique 1 To pertorm the analysis select Other and then Comparative quantitation in the Analysis window Double click on the channel to analyze Warton K Foster N C Gold W A and Stanley K K 2004 A novel gene family induced by acute inflammation in endothelial cells Gene 342 85 7 44 Rotor Gene Q User Manual 11 2012 Analysis User Interface 2 Choose a control sample using the dro
40. in the correct position a warning beep sounds Rotor Gene Q User Manual 11 2012 Operating Procedures Hardware 9 When sealing is finished a beep sounds and the orange Ready light illuminates Press down on the blue anodized bar to raise and lock the sealing mechanism back in its original position Do not continue sealing for any longer than indicated by the beep or the Rotor Disc may deform 10 Slide the Rotor Disc Loading Block out of the Rotor Disc Heat Sealer Allow the film to cool for approximately 10 seconds and then gently remove the excess film 11 Remove the Rotor Disc from the Rotor Disc Loading Block 12 Load the Rotor Disc into the rotor using the position one locator tab as a guide to the correct orientation 5 12 Rotor Gene Q User Manual 11 2012 Operating Procedures Software 6 1 Operating Procedures Software New runs can be set up using the Quick Start wizard or the Advanced wizard which appear when the software is started up The Quick Start wizard is designed to allow the user to start the run as rapidly as possible The Advanced wizard enables more options such as configuration of Gain Optimisation and volume settings For convenience the wizards have a number of templates with default cycling conditions and acquisition channels To change the wizard type select the appropriate tab at the top of the New Run window Quick Start wizard The Quick Start wizard allow
41. kl ou can pause the run an this screen A E marker will be recorded in the channel data to shaw the duration of the pause Resume Help Close WARNING Hot surface W18 When pausing a run the Rotor Gene Q will not be cooled completely to room temperature Exercise caution before handling the rotor or any tubes in the instrument 7 7 3 Stop Run If this option is selected a prompt will appear asking for confirmation that the run should be stopped 7 8 View menu 7 8 1 Run Settings General This window allows the setup of run information run filename analysis date operator and any associated notes 7 66 Rotor Gene Q User Manual 11 2012 Analysis User Interface The window contains all information except for the profile required to configure a run After a run has finished the following information is displayed in this window cycler used gain settings number of channels and time of start and finish General Machine Options Messages Channels Tube Layout Security Filename FD epartments L E xpenmentalD ata AG SEA G BOCDOSRH 1 2052 5D ark Operator Motes E Reaction Volume Other Aun Information 20 This refers to the total volume of the mixture x in the tube Channels saved for this run Cycling 4 Green Cycling B r ellew Cycling C Orange Cycling D Hed Created with template C Documents and Settingsslabpc CORBET T Desktop Tem
42. l utilisateur et non de s y substituer Rotor Gene Q User Manual 11 2012 B 1 Appendix B 1 1 Utilisation appropri e AVERTISSEMENT Risque d accident corporel et de d t rioration ATTENTION du mat riel 1 L utilisation inappropri e du Rotor Gene Q peut entra ner des accidents corporels ou une d t rioration de l appareil Le Rotor Gene Q ne doit tre utilis que par du personnel qualifi ayant t convenablement form L entretien du Rotor Gene Q doit tre effectu uniquement par des sp cialistes de l entretien sur site QIAGEN Proc der la maintenance comme d crit dans la Section 9 QIAGEN facture les r parations rendues n cessaires suite une maintenance inappropri e AVERTISSEMENT Risque d accident corporel et de d t rioration ATTENTION du mat riel W2 Le Rotor Gene Q est un appareil lourd Afin d viter tout accident corporel et toute d t rioration de l appareil faites attention en le soulevant AVERTISSEMENT Risque d accident corporel et de d t rioration ATTENTION du mat riel w3 est en marche A N essayez pas de d placer le Rotor Gene Q lorsqu il ATTENTION D t rioration de l appareil C1 vitez de renverser de l eau ou des produits chimiques sur le Rotor Gene Q La d t rioration due de l eau o des produits chimiques renvers s annulera votre garantie Remarque en cas d urgence teignez le Rotor Gene Q l aide de l interrupteur
43. m Reports Arrange Smart Tiling Tile Horizontally Tile Vertically Cascade Toolbar These buttons are shortcuts to frequently used operations These operations can also be accessed from the drop down menus P d New Open Save View ru r Wl Py S Settings Progress Profile Temp Samples Analysis Reports Arrange gt i Start Pause Stop e Help channels 2 Cycling A Green 2 Cycling A Yellow 2 Cycling 4 Orange Cycling A Red View raw channels Click on these buttons to view the raw unanalyzed data from particular channels in the run Channels 2 Cycling A Green 2 Cycling A Yelow 2 Cycling 4 Orange 2 Cycling A Red Rotor Gene Q User Manual 11 2012 7 1 Analysis User Interface When viewing this data a number of options are available to change the data presentation The raw data may also be transformed to facilitate different types of analysis Adjust Scale To select Adjust Scale click the right mouse button over the appropriate window Adjust Scale brings up a window in which a scale can be specified Adjust Scale Maximum 10 4 Minimum 4 Hs EE Autoscale Autoscale attempts to fit the scale to the maximum and minimum readings of the data Default Scale Default Scale resets the scale to display from O to 100 fluorescence units Spanner wrench See Section 8 5 for more information ICON
44. region at the beginning of the run represents the calibration process The green dots represent the acquisitions taken each cycle during heating The blue dots represent the acquisition at the end of the anneal step at 60 C Note that while the profile shows each step with the same denature temperature this may not be the case If the sample requires slightly longer to melt towards the end of the run the optical denature process waits for the melt according to the fluorescent data and not according to time For this reason the temperature trace may vary for each cycle 2 Click on the first half of the graph with the Optical Denature symbol x The Calibration Settings information appears on the left of the screen This cycle repeats 45 time s Click on one of the steps below to modify it or press or to add and remove steps for this cycle Optical Denature Calibration Settings Acquiring to RealTime on tube 1 Ramp from 80 to 95 and hold for 3 mins 0 secs 60 deg for 40 secs OK 3 The Calibration Settings information is usually correct To modify it if necessary click Edit The Calibration Settings window appears Rotor Gene Q User Manual 11 2012 6 2 Operating Procedures Software I Calibration Settings Settings The Optical Denature Calibration settings determine how to perform the initial melt up to the denaturation temperature ta determine the fluorescence level
45. reliable analysis Samples spin continually at 400 rpm during a run Centrifugation prevents condensation and removes air Rotor Gene Q User Manual 11 2012 3 1 General Description bubbles but does not pellet DNA In addition samples do not need to be spun down prior to a run Samples are heated and cooled in a low mass air oven Heating is achieved by a nickel chrome element in the lid The chamber is cooled by venting the air out through the top of the chamber while ambient air is blown up through the base Heating Heater Centrifugal fan drives elements air around chamber ewiteh on Im m Chamber vent seals jp SP ver P s FF to contain air Serr ery AA EL Cooling e Heater e gpuruga ran drives elements air around chamber switch off Chamber vent opens expelling hot air Centrifugal fan Orves air into chamber Cool air in Illustration of the heating and cooling system 322 Rotor Gene Q User Manual 11 2012 General Description 3 2 Optical system With a choice of up to 6 excitation sources and 6 detection filters combined with a short fixed optical path the Rotor Gene Q can be used for multiplex reactions ensuring minimum fluorescence variability between samples and eliminating the need for calibration or compensation Samples are excited from the bottom of the chamber by a light emitting diode Energy is transmitted through the thin walls at the base of the tube Emit
46. reported for calculated concentrations rather than an absolute value This is a percentage variation For example a value of 1 07537 represents a 7 54 variation 278 974 322 611 2 300 000 1 07537 300 000 1 07537 Reporting an absolute value does not make sense for a standard curve The value could be reported at the lowest concentration to create a perceived low error 3 copies or at the high concentration 3 000 000 copies For this reason the Orders of Magnitude standard deviation is reported E For calculated concentrations the geometric mean is used instead of the arithmetic mean This accounts for the exponential nature of real time PCR For example in the case of twofold dilutions with 1 2 8 and 16 copies the average should be 4 copies because it is the middle of the dilution series However the arithmetic mean is 6 75 The geometric mean is 1 2 8 16 1 4 4 copies Rotor Gene Q User Manual 11 2012 7 25 Analysis User Interface Dynamic tube normalization The Dynamic Tube option is selected by default and is used to determine the average background of each sample just before amplification commences Standard normalization simply takes the first 5 cycles and uses these as an indicator of the background level of each sample All data points for the sample are then divided by this value to normalize the data This can be inaccurate because for some samples the background level over the first 5
47. select Other and then Scatter Graph Analysis in the Analysis window When performing scatter graph analysis it is not sufficient to double click on one channel to analyze because this analysis is performed using 2 channels simultaneously To perform this analysis either hold down SHIFT and click to highlight the channels to analyze or drag the mouse pointer over the channels Once the desired channels have been highlighted click Show The list will update to show all the channels on one line with a checkmark next to them This indicates that they will all be used in one analysis To remove one or more of these channels right click on the analysis and select Remove Analysis Those channels can then be included in another scatter graph analysis A channel can only be used in one analysis at a time Reports This opens the Scatter Analysis report for preview Results This displays the Scatter Analysis Results window The genotype for each sample is determined by the regions defined by the user on the scatter graph Normalization A variety of options are available to options optimize the way in which the raw data plots are normalized E Dynamic Tube dynamic tube normalization M Slope Correct noise slope correction M Ignore First x cycles correction for noise in initial cycles For more details see page 7 25 7 50 Rotor Gene Q User Manual 11 2012 Analysis User Interface Genotype
48. software 6 Connect the USB cable or RS 232 serial cable supplied to a USB or communications port on the back of the computer 7 Connect the USB or RS 232 serial cable to the back of the Rotor Gene Q 8 Connect the Rotor Gene Q to the power supply Connect one end of the AC power cord to the socket located at the rear of the Rotor Gene Q and the other end to the AC power outlet NN On off switch EE Cli amodo Power supply port tom m Type plate including Cooling fan Serial port USB port serial number Rotor Gene Q User Manual 11 2012 4 5 Installation Procedures 4 7 4 6 Note Only connect the Rotor Gene Q to the computer with the USB and serial cables delivered with the instrument Do not use other cables Software installation 1 To install the Rotor Gene Q software insert the CD software delivered with the instrument into the CD drive of the computer 2 Select Install Operating Software in the window that appears Note For easy installation please refer to the Rotor Gene Q Installation Guide provided with the instrument to guide you through the next steps of software installation Rotor Gene Q Pure Detection Install Operating Software Exit Sample amp Assay Technologies Rotor Gene Q User Manual 11 2012 Installation Procedures 3 Once the sofiware has been installed a desktop icon will be created automaticall
49. the software and the serial number and model of the instrument Rotor Gene Q User Manual 11 2012 4 9 Installation Procedures Rotor Gene Q Pure Detection Software Version 2 2 2 Build 8 Machine Serial No 090839 Model 5 Plex HRM Updates and additional information available at http www qiagen com Warning This computer program is protected by copyright law and international treaties Unauthorized reproduction of this program or any portion of it may result in severe civil and criminal penalties and will be prosecuted to the maximum extent possible under the law Portions of Info Zip c 1990 1999 Info ZIP Pty Ltd i System Info Help Sample amp Assay Technologies The software may be freely copied for use within an organization that owns a Rotor Gene Q The software may not be copied and distributed to others outside the organization 4 9 Additional software on connected computers Rotor Gene Q software manages time critical processes during the PCR run and the data acquisition process For this reason it is important to ensure that no other processes use significant system resources and thus slow down the Rotor Gene Q software It is particularly important to pay attention to the points listed below System administrators are advised to consider any impact that a modification to the system may have on the resources before implementing it 4 10 Rotor Gene Q User Ma
50. to correct the interference by one or more of the following measures Rotor Gene Q User Manual 11 2012 Appendix A M Reorient or relocate the receiving antenna MH Increase the separation between the equipment and receiver M Connect the equipment into an outlet on a circuit different from that to which the receiver is connected Consult the dealer or an experienced radio T V technician for help QIAGEN GmbH Germany is not responsible for any radio television interference caused by unauthorized modifications of this equipment or the substitution or attachment of connection cables and equipment other than those specified by QIAGEN GmbH Germany The correction of interference caused by such unauthorized modification substitution or attachment will be the responsibility of the user Rotor Gene Q User Manual 11 2012 A 5 Appendix A Declaration of Conformity Declaration of Conformity Name and address of the company QIAGEN GmbH QIAGEN Strasse 1 D 40724 Hilden Germany We herewith declare that the product Rotor Gene Q Accessories Rotor Gene Heat Sealer meets all applicable requirements of the following European Directives Low Voltage Directive LVD 2006 95 EC Electromagnetic Compatibility Directive EMC 2004 108 EC and the relevant harmonised standards IEC 61010 1 Ed 2 IEC 61010 2 010 Ed 2 IEC 61010 2 081 Ed 1 IEC 61326 1 IEC 61000 6 2 IEC 55022 Hombrechtikon 04 November 2009 Pit Mug
51. useful for determining correlation coefficient M and B The slope M and the intercept B of the standard curve are automatically calculated using the formula y Mx B and shown in the Standard Curve window Export Graph Clicking the right mouse button over the standard curve displays the option to export the graph see Section 8 4 Overlay When multiple quantitation runs have been performed in the same run it is possible to overlay the standard curves in the same window This is useful for graphically viewing the difference between different thresholds This feature is shown in the screenshot below Rotor Gene Q User Manual 11 2012 7 15 Analysis User Interface E Standard Curve Cycling A Green Page 1 nmi x Cycling A Green Page 11 F 0 99969 F 2 0 99939 3 292 B 38 843 Efficiency 1 01 Export Graph Standard Error Mean Cycling 4 Yellow Page 27 w Show Stats Cycling Orange Page 3 Zoom CILE Cycling A Red Page 4 Return bo Default Position PL l 1 1000 1001 10 02 10 03 1004 1005 Concentration Standard curve calculation conc C and are 2 versions of the equation used to relate values and concentrations In publications the formula is most commonly used The standard curve can be either Floating or Fixed If Floating an optimal equation for the standard curve is calculated each
52. 0 Rotor Gene Multiplex PCR Master Mix 2x2 ml RNase Free Water Rotor Gene Multiplex For 400 x 25 ul reactions 3x 1 7 ml 2x 204974 RT PCR Kit 400 Rotor Gene Multiplex RT PCR Master Mix 100 ul Rotor Gene RT Mix 2 x 2 ml RNase Free Water QuantiTect Virus For 200 x 50 ul reactions QuantiTect 211033 ROX Vial Kit 200 Virus NR Master Mix without ROX dye ROX Dye Solution QuantiTect Virus RT Mix RNase Free Water QuantiTect Nucleic Acid Dilution Buffer QuantiTect Primer For 200 x 50 ul reactions or 400 x 25 ul Varies Assay 200 reactions 10x QuantiTect Primer Assay lyophilized supplied in single tube For information about additional kit sizes and additional kits visit www qiagen com Rotor Gene Q User Manual 11 2012 E 5 Appendix E Product Contents Cat no QuantiTect Rev For 50 x 20 ul reactions gDNA Wipeout 205311 Transcription Kit 50 Buffer Quantiscript Reverse Transcriptase Quantiscript RT Buffer RT Primer Mix and RNase Free Water miScript SYBR Green For 200 reactions QuantiTect SYBR 218073 PCR Kit 200 Green PCR Master Mix miScript Universal Primer Type it Fast SNP For 100 x 25 ul reactions 2x Type it Fast 206042 Probe PCR Kit 100 SNP Probe PCR Master Mix 5x Q Solution RNase Free Water Type it HRM PCR Kit For 100 x 25 ul reactions 1 x 1 3 ml of 206542 100 2x HRM PCR Master Mix contains HotStarTaq Plus DNA Polymerase EvaGreen dye optimized concentration of Q
53. 0 7 72 05 228 10 38 0 09 10 15 10 60 BOE ofS 2 621 05 2 Oing Green Page 1 A Green Page 1 Cycling Givon Page 1 10 13 49 05 SF 3 2 5 0 13 1331 1358 BTE 4 DE Cycling A Green 1 Cycling A Gisan Page 1 12 52x Cycling Green Page 1 Stand 556 22 1227 1546 025 14 04 10 00 258 04 7 021 02 804 Cycing Green Page 1 J Cycing A Green Page 1 15 Oing A Green Page 1 16 Silor 1 E D NEAR 218 024 20 43 21 3 LEE 02 9 1 2E 401 031 Gieen Page 1 17 Ba Cycling Gieen Page 1 18 Cycling Gisan Page 1 19 2 NEG Mus Cu 1008 00 Cycling A Green Page 1 20 2 Stand 2156 10400 760 Cycling Green Page 1 2 2 Stand Hu 1000 02 Cycing A Green Page 1 22 im rc NEG INTC A Green Page 1 23 din c NEG INTC Cocina A Green Page 1 2 C NEG NTC In the Quantitation Results window the results from the run are summarized in a table Clicking the right mouse button and selecting Export to Excel exports the table to Excel Excel opens automatically To copy the data into an existing spreadsheet choose the Copy option instead open the spreadsheet then select Paste 7 20 Rotor Gene Q User Manual 11 2012 Analysis User Interface The Quantitation Results window includes the following columns Analysis No Colour Type Ct Ct Comment The current data set acquiring chan
54. 1 14 Important In order to provide accurate reporting and traceability if a C value is excluded automatically the Ct Comment column should not be hidden at any time The Ct Comment column must be activated manually after the first software installation or if a previous user has deactivated this column For increased convenience the AutoStat feature automatically calculates the average standard deviation and minimum and maximum values of samples of interest Select the results of interest by dragging with the left mouse button and the values are displayed in a table on the right of the screen Rotor Gene Q User Manual 11 2012 Analysis User Interface In this screenshot the concentrations of several samples are analyzed Quant Results Cycling A Green Page 1 Mil Statistics Masimum 20730050 Minimum 25142920 30000000 Court 30000000 320 30000000 28730060 Mean 27328521 3000000 Std Dev 1 07537 3000000 3103391 Orders of Mag 3000000 3457111 300000 285393 300000 298898 300000 275802 Copy m JAE IMPORTANT The AutoStat feature is context aware This means that where possible it only generates information that is useful For example It is not possible to obtain 95 confidence interval from a set of selected calculated concentrations because the regression model must also be taken into account M The Orders of Magnitude standard deviation is
55. 1 2012 1 Appendix A Transportation conditions Air temperature 25 C to 60 C 13 F to 140 F in manufacturer s package Relative humidity Max 75 noncondensing Environmental 2K2 IEC 60721 3 2 class Storage conditions Air temperature 15 C to 30 C 59 F to 86 F in manufacturer s package Relative humidity Max 75 noncondensing Environmental 1K2 IEC 60721 3 1 class Mechanical data and hardware features Dimensions Width 370 mm 14 6 in Height 286 mm 11 3 in Depth without cables 420 mm 16 5 in Depth door open 538 cm 21 2 in Weight 12 5 kg 27 6 lb standard configuration Capacity Up to 100 samples per run using a Rotor Disc 100 Software Rotor Gene Q software version 2 1 0 or higher supplied on the installation CD provided A 2 Rotor Gene Q User Manual 11 2012 Appendix A Thermal specifications Description Temperature range Temperature accuracy Temperature resolution Temperature uniformity Optical specifications Description Excitation sources Detector Acquisition time Rotor Gene Q User Manual Specification 35 C to 99 C 95 F to 210 2 F 0 5 C 0 02 C smallest programmable increment 0 02 C standard deviation Specification High energy light emitting diodes Photomultiplier 4s 11 2012 A 3 Appendix A FCC Declaration The United States Federal Communications Commission USFCC in 47 CRF 15 105 declared th
56. 2 Rotor Gene Q User Manual 11 2012 Analysis User Interface x Quick Start Advanced T L Perform Last Run concentration of Three Step with Melt nucleic acid using intercalating dyes ae i h Two Step oe Quenched FRET lucleic Add Concentration Measurement Jj HRM Other Runs Open A Template In Another Folder Cancel Help lv Show This Screen when Software Opens Note Ensure that a positive control such as a high concentration standard is run in tube position 1 Without a positive control the software will be unable to optimize gain settings for maximum sensitivity You will be prompted for this before each run Analysis Concentration analysis operates by relating the fluorescence level to a concentration value Two analysis models are available The optimal analysis to chose depends on the chemistry and application Linear Regression analyzes data by assuming a linear relationship and estimating unknown values on the basis of a generated linear model It determines measurement error by examining the deviation of the readings from a linear model Rotor Gene Q User Manual 11 2012 7 63 Analysis User Interface 7 6 11 If concentration readings are linear this is the most suitable analysis because it provides statistical analysis of variation ANOVA to the user Spline Curve Fit assumes only that concentrati
57. 2 Well Rotor The 72 Well Rotor is blue in color The 72 Well Rotor and 72 Well Rotor Locking Ring are used with Strip Tubes and Caps 0 1 ml which can be used for volumes as low as 10 ul The caps provide a safe and reliable seal Rotor Disc 72 Rotor The Rotor Disc 72 Rotor is dark gray in color The Rotor Disc 72 Rotor and Rotor Disc 72 Locking Ring enable use of the Rotor Disc 72 The Rotor Disc 72 is a disc with 72 wells for high throughput use To seal the Rotor Disc 72 a clear polymer film is applied to the top and heat sealed The film is quick to apply and prevents contamination by providing a Rotor Gene Q User Manual 11 2012 Operating Procedures Hardware strong durable and tamper proof seal For more information on the Rotor Disc 72 see Section 5 3 Rotor Disc 100 Rotor The Rotor Disc 100 Rotor is gold in color The Rotor Disc 100 Rotor and Rotor Disc 100 Locking Ring enable use of the Rotor Disc 100 The Rotor Disc 100 is a disc with 100 wells for high throughput use The Rotor Disc 100 is the rotary equivalent of a 96 well plate but with an additional 4 reference wells It enables integration of the Rotor Gene Q with 96 well laboratory workflows The extra wells can be conveniently used for more samples additional control reactions or orientation reactions without occupying any of the standard 96 well positions For seamless 96 well workflow compatibility Rotor Disc 100 wells use 96 well plate labelin
58. 2012 Operating Procedures Software reference tube must also contain a detection chemistry that enables detection of strand dissociation When heating to the initial denature temperature a melt is performed on the green channel from 80 C to 95 C by default The parameters of this initial melt can be adjusted by the user From this data a melt curve is generated and automatically analyzed 4 75 80 85 90 95 10C c The melt peak is referenced back to the raw data to obtain a denature threshold Then every Optical Denature Cycling step the instrument is heated as quickly as possible and data is acquired continually Once the reference tube has reached the denature threshold fluorescence level the instrument is immediately cooled and proceeds to the next programmed step in the cycle A peak is not calculated while cycling Instead the fluorescence level is referenced to the melt peak and this designates the denature threshold In the tollowing graph the raw fluorescence readings and the first derivative have been overlaid This shows the correspondence between the denature threshold and the melt peak obtained during the calibration Rotor Gene Q User Manual 11 2012 6 19 Operating Procedures Software Denature threshald 75 8D 85 ap ec To perform Optical Denature Cycling you will need M A preamplified PCR product to place in position 1 of the rotor This sample should contain the same
59. 3 E as ce Quantitative methylation analysis by HRM Various ratios of methylated and unmethylated DNA APC adenomatosis polyposis coli were analyzed and discriminated by HRM methylation analysis on the Rotor Gene Q using the EpiTect HRM Kit a standard normalized melt curve and BR difference plot normalized to the 50 methylated sample are shown 11 6 Rotor Gene Q User Manual 11 2012 High Resolution Melt Analysis 11 5 Guidelines for successful HRM analysis The success of HRM analysis depends largely on the particular sequence under investigation Certain sequence motifs such as hairpin loops or other secondary structures localized regions of unusually high or low GC content or repeat sequences can all affect the outcome In addition use of standardized kits and optimized protocols from QIAGEN can overcome many of the potential challenges listed Some simple guidelines to help ensure success are detailed below Analyze small DNA fragments Analyze fragments no greater than about 250 bp Larger products can be analyzed successfully but usually provide lower resolution This is because for example a single base variation has a greater effect on the melting behavior of a 100 bp amplicon than on a 500 bp amplicon Ensure that PCR contains only specific product Samples contaminated with post PCR artifacts such as primer dimers or nonspecific products can make HRM results difficult to interpret Kits from QIAGEN for HRM
60. 384 well LightScanner Idaho Technology National Genetics Reference Laboratory Wessex Rotor Gene Q User Manual 11 2012 11 3 High Resolution Melt Analysis Heterozygote Homozygote mutant 11 4 Rotor Gene Q User Manual 11 2012 High Resolution Melt Analysis Confidence 44 Human SNP 60051276 homo unknown homo unknown homo B AG Human SNP 60091276 hetero AG unknown hetero unknown hetero Al Bl GG Human SNP 60021276 homo GG unknown hama GG unknown homo Go SNP genotyping by HRM Human SNP rs60031276 Ato G substitution in the PPP1R14B gene protein phosphatase 1 regulatory inhibitor subunit 14B was analyzed on the Rotor Gene Q using 10 ng genomic DNA of different genotypes and the Type it HRM Kit Homozygous wild type AA homozygous mutant GG and heterozygous AG samples are shown on A a standard normalized melt curve and BR difference plot normalized to wild type samples Genotypes for the unknown samples were assigned by the Rotor Gene Q software 11 4 Methylation analysis example In the example shown the EpiTect HRM PCR Kit was used in HRM analysis to discriminate various ratios of methylated and unmethylated DNA For technical details consult the EpiTect HRM PCR Handbook Rotor Gene Q User Manual 11 2012 11 5 High Resolution Melt Analysis A 100 methylated ras ras rec res 2 TT 3 Tas 322 ars as sc 2
61. 7 30 import 7 16 overlay 7 15 two standard curve method 7 31 Storage A 2 Suitabilities 7 80 Support 7 104 Symbols 1 10 T Technical assistance 2 2 TeeChart Office 8 4 8 7 Temperature graph 7 71 Templates adding to advanced wizard 6 9 adding to quick start wizard 6 3 allelic discrimination 7 49 8 1 endpoint analysis 7 61 8 1 melt analysis 7 44 8 1 quantitation 7 31 8 1 scatter graph analysis 7 52 8 1 Thermal performance 3 1 Three step with melt 6 2 6 8 Threshold 7 19 Toggler 7 3 Toolbar 7 1 Touchdown 6 15 Transportation A 2 Troubleshooting 12 1 HRM 12 1 log archives 12 1 Rotor Gene Q 12 3 Tube layout 7 69 Two standard curve method 7 31 Two step 6 2 6 9 Rotor Gene Q User Manual 11 2012 Index 3 Index U V Unpacking 4 3 Version 2 2 User Virtual mode 4 9 7 10 assigning roles 7 90 creating account 7 86 multiple accounts 7 95 W Warnings 1 1 Waste disposal 1 7 Wrench icon 8 6 Index 4 Rotor Gene Q User Manual 11 2012 www giagen com Australia techservice au giagen com Austria techservice at qiagen com Belgium techservice bnl8qiagen com Brazil suportetecnico brasil giagen com Canada techservice ca giagen com China techservice cn giagen com Denmark techservice nordic giagen com Finland techservice nordic qiagen com France techservice fr giagen com Germany techservice de giagen com Hong Kong techservice hk giagen com India techse
62. 95 deg Hold Time 3 mins 0 secs If performing Optical Denature Cycling a Hold can be used as a calibration step In this case a calibration melt is performed before the Hold By default this is configured for the first Hold in the run but may be changed if required Rotor Gene Q User Manual 11 2012 6 13 Operating Procedures Software Click on a cycle below to modify it Cycling Hold Temperature gr dea Hold Time 3 mins 0 secs Calibration Step M Calibration Settings Acquiring to an tube 1 Ramp from 80 to 35 and hold for 3 mins secs Offset 0 deg E dit For more information about Optical Denature Cycling see page 6 18 Cycling Cycling repeats the user defined temperature and time steps a specified number of times The number of repeats is set using the This cycle repeats X time s button A single cycle is displayed graphically as shown in the screenshot below Each step of the cycle can be altered The temperature can be changed by dragging the temperature line in the graph up or down The duration of the step can be changed by dragging the temperature boundary in the graph left or right Alternatively click on the step and use the temperature and time buttons to the left of the graph 6 14 Rotor Gene Q User Manual 11 2012 Operating Procedures Software Steps can be added or removed from the cycle using the and but
63. AM ar En 2 2 H 7 The implication of this formula is that adding replicates to an standard individual concentration reduces the width of the interval for all estimates as n is increased Adding a large number of replicates to an unknown reduces its uncertainty to that of a single standard The extra replicates reduce the uncertainty due to the unknown not forming part of the linear model Confidence intervals for C values We assume that error in replicate C values is linear and normally distributed We therefore use the One Sample t Confidence Interval Let be the mean value for a replicate s values Tg In 1 Then a 100 1 a confidence interval for a value u is TT Le 4 1 V V Fi We would like to thank Peter Cook from the Mathematics Department of the University of NSW Sydney Australia whose help was invaluable in verifying the mathematical approaches used ul D 2 Rotor Gene Q User Manual 11 2012 Appendix E Appendix E Rotor Gene Q products accessories and consumables Product Rotor Gene Q 2plex Rotor Gene Q 2plex HRM Rotor Gene Q 5plex Rotor Gene Q 5plex HRM Rotor Gene plex RGQ Basic Agreement no PM Contents Cat no Real time PCR cycler with 2 channels Inquire green yellow laptop computer software accessories 1 year warranty on parts and labor Real time PCR cycler and High Resolution Inquire Melt
64. All Start Manual Changing Gain During a Run Remove removes the highlighted channel Remove All removes all channels Start begins Gain Optimisation gain is chosen which results in fluorescence signal levels within the specified range If fluorescence falls outside the specified range the gain is set to give the closest match possible This opens the Manual Gain Adjustment window see page 6 29 If the gain at the beginning of the run was too high or too low it can be changed within the first ten cycles A vertical line appears where the gain has been changed The cycles before the change are excluded from the analysis Note Gain Optimisation may chose a setting which does not fall within the specified range This can be due to changes in fluorescence after the first Hold step However the result of Gain Optimisation provides a good indication of the fluorescence level the run will be started on 6 28 Rotor Gene Q User Manual 11 2012 Operating Procedures Software Manual Gain Adjustment To perform Manual Gain Adjustment click on Manual in the Auto Gain Optimisation Setup window The Manual Gain Adjustment window appears This window displays the fluorescent readings at any given temperature in real time It is used when the background of a sample is unknown and therefore the gain must be determined to ensure the sample signal is sufficient for detection File Help
65. B 10 Rotor Gene Q User Manual 11 2012 Appendix B 1 9 Maintenance Proc dez la maintenance comme d crit dans la Section 9 QIAGEN facture les r parations rendues n cessaires suite une maintenance inappropri e AVERTISSEMENT Risque d accident corporel et de d t rioration ATTENTION du mat riel W19 N effectuez que la maintenance sp cifiquement d crite dans ce manuel d utilisation AVERTISSEMENT Risque d incendie W20 En nettoyant le Rotor Gene Q avec un d sinfectant alcoolis laissez le couvercle du Rotor Gene Q ouvert pour laisser les vapeurs inflammables se disperser Ne nettoyez le Rotor Gene Q qu une fois que la chambre a refroidi AVERTISSEMENT Risque de d charge lectrique ATTENTION Ne d montez pas l appareil Rotor Gene Q ATTENTION D t rioration du boitier de l appareil C8 Ne nettoyez jamais le boitier de l appareil avec de l alcool ou des solutions base d alcool L alcool endommagera le boitier N utilisez que de l eau distill e pour nettoyer le boitier Rotor Gene Q User Manual 11 2012 B 11 Appendix B 1 10 Symboles du Rotor Gene Q Danger li la chaleur la Pr s de la chambre temp rature de la chambre type visible quand le peut atteindre des couvercle est ouvert temp ratures sup rieures 120 C 248 F Lire les informations dans le Arri re de l appareil manuel Plaque signal tique Symbole CE pour l arri re de l appareil Communaut
66. Cycling Changing an existing step to use Optical Denature Cycling To change an existing Denature step in a cycling sequence select the cycle in the list in the Edit Profile window Then select the Denature step by clicking on it in the display 60 deg for 40 secs DK 95 deg for 20 secs Not Acquiring Long Range Touchdown Click on the drop down menu and select Optical Denature The temperature and hold time are removed and the Optical Denature icon is displayed Rotor Gene Q User Manual 11 2012 6 23 Operating Procedures Software Gain Optimisation When setting up a new run it is helpful to use the Gain Optimisation function This allows you to optimize the gain to a setting that will provide the desired range of starting fluorescence at a set temperature usually the temperature at which data acquisition occurs in each of the channels being acquired The aim of Gain Optimisation is to ensure that all data is collected within the dynamic range of the detector If the gain is too low the signal will be lost in background noise If it is too high all signal will be lost off scale saturated The gain range for each channel is 10 to 10 where 10 is the least sensitive and 10 is the most sensitive When running reactions for the first time we recommend preparing a test sample containing all the reaction components Place the test sample in the Rotor Gene
67. Digital Filter 8 6 See Section 8 3 This saves the graph in a variety of formats see Section 8 4 This copies the graph image to the clipboard This opens the graph directly in TeeChart Office for editing see Section 8 4 This prints the graph This modifies the currently selected digital filter on the graph The digital filter smoothes data using a sliding window of points Rotor Gene Q User Manual 11 2012 Additional Functions 8 6 Select digital filter for graph Cancel Show Pinpointer This opens a window that displays the exact coordinates of the mouse pointer position Grouping This visually groups samples that have identical names This can be useful on full rotor runs Selecting this option does not affect calculated values Selected area options An area of a graph can be selected by clicking and holding the left mouse bution and dragging the mouse pointer The following options appear Select Only These Samples Deselect These Samples Zoom Cancel Select Only Samples outside the selected area are These Samples deselected Deselect These All samples in the selected area are Samples deselected Zoom This zooms in on the selected area of the graph Click the Default Scale button to ZOOM out Rotor Gene Q User Manual 11 2012 8 7 Additional Functions This page intentionally left blank 8 8 Rotor Gene Q User Manual 11 2012 Maintenance Proced
68. November 2012 Rotor Gene Q User Manual Rotor Gene Q QIAGEN Trademarks QIAGEN QlAgility EpiTect HotStarTaq HRM Quantiscript QuantiTect Rotor Gene Rotor Disc Type it QIAGEN Group CAL Fluor Quasar Biosearch Technologies Inc Cy GE Healthcare EvaGreen Biotium Inc LC Green Idaho Technology Inc Alexa Fluor FAM JOE Marina Blue SYBR SYTO Texas Red VIC Life Technologies Corporation Yakima Yellow Nanogen Inc LightCycler Roche Group Core Intel Intel Corporation Adobe Illustrator Adobe Systems Inc Microsoft Windows Excel Microsoft Corporation Registered names trademarks etc used in this document even when not specifically marked as such are not to be considered unprotected by law TeeChartOffice Copyright 2001 2002 by David Berneda All rights reserved For applicable countries This real time thermal cycler is licensed under pending U S Patent rights for an apparatus or system covering automated thermal cyclers with fluorescence detectors and seeking priority to U S Serial No 07 695 201 and corresponding claims in any foreign counterpart patent thereof owned by Applied Biosystems LLC in all fields including research and development all applied fields and human and animal in vitro diagnostics No rights are conveyed expressly by implication or estoppel to any patents on real time methods
69. On All Ott Edit Samples 7 4 Rotor Gene Q User Manual 11 2012 Analysis User Interface 7 5 7 5 1 Rotor Gene Q User Manual Page Toggle Sample ID Display This label at the top of the toggler indicates the sample page tha is displayed Pages allow varied independent analyses from one channel data set For example you can run two standard curves in the green channel and generate independent reports More information on setting up sample pages is available in Section 7 8 4 If a 72 Well Rotor is used samples are shown in the format A1 to A8 B1 to B8 etc The Toggle Sample ID Display option allows the user to switch to a numerical sample order 1 to 72 Select Non Empty This option deselects any samples that have Samples Select Groups File menu New a Type specified as None in the Edit Samples window This ensures that only samples relevant for the analysis are displayed If you have defined groups this feature will toggle switch on off the display of the samples in the groups Groups are arbitrary collections of samples that allow advanced reporting of statistical results For example groups of treated and untreated patient samples can be defined Groups can be set up in the Edit Samples window After selecting File and then New the New Run window appears This window provides commonly used templates organized under Quick Start and Advanced tabs Once the
70. R ckseite des Rotor Gene Q und der Raumwand eingehalten wird damit eine ausreichende Bel ftung des Ger ts gew hrleistet ist Die L ftungsschlitze und ffnungen die f r die Be und Entl ftung des Rotor Gene Q bestimmt sind d rfen nicht verdeckt werden 1 8 berhitzungsgefahr WARNUNG Hei e Oberfl chen W17 In der Rotorkammer des Rotor Gene Q k nnen Temperaturen von ber 120 C erreicht werden Ber hren Sie sie nicht wenn sie aufgeheizt ist WARNUNG Hei e Oberfl chen W18 Wenn ein Lauf unterbrochen wird k hlt sich der Rotor Gene Q nicht vollst ndig auf Raumtemperatur ab Seien Sie entsprechend vorsichtig wenn Sie den Rotor oder eines der Reaktionsgef e im Ger teinneren ber hren C 10 Rotor Gene Q User Manual 11 2012 Appendix C 1 9 Wartungsarbeiten F hren Sie alle Wartungsarbeiten gem den Anweisungen in Abschnitt 9 durch QIAGEN stellt alle Reparaturen in Rechnung die nachweislich auf eine inkorrekte Wartung zur ckzuf hren sind WARNUNG Verletzungsgefahr und Besch digung des Ger ts wis ACHTUNG F hren Sie nur Wartungsarbeiten durch die ausdr cklich in diesem Handbuch beschrieben werden WARNUNG Brandgefahr W20 Lassen Sie beim Reinigen des Rotor Gene Q mit einem Desinfektionsmittel auf Alkoholbasis den Deckel ge ffnet damit sich entflammbare D mpfe verfl chtigen k nnen Reinigen Sie den Rotor Gene Q nur wenn sich die Rotorkammer abgek hlt hat
71. R 1000 Standard 1 000 Yes R100 Standard 100 Yes 4 R100 Standard 100 Yes 10 Standard 10 Yes AB R10 Standard 10 Yes A R1 Standard 1 Yes R1 Standard 1 Yes B1 RNTC NTC Yes B2 RNTC NTC Yes Hio 11000 Standard 1 000 Yes B 11000 Standard 1 000 Yes Page Name Page 1 4 gt New Delete Synchronize pages Undo OK Cancel Help Click on the Samples button to bring up the Edit Samples window The Edit Samples window can also be accessed by right clicking over the sample list on the right of the screen This window has identical functionality to the Edit Samples window in the wizards except that the toolbar functions are also available in the File and Edit menus Four menus appear at the top of the window File Edit Format and Security The File menu is used to create a new blank Edit Samples window to open an existing sample template or to save sample names as a template for future use The extension of these template files is smp The Edit menu allows rows to be copied and pasted The Security menu enables the sample definitions to be locked Rotor Gene Q User Manual 11 2012 7 73 Analysis User Interface Given Conc Format Unit Line style Edit Reset Default Gradient Ci ICOn gm This drop down menu is used to choose a suitable format for the concentration display Concentrations are automatically
72. Rotor Gene Q User Manual 11 2012 Analysis User Interface 4 Enter your e mail account name and password if your server uses secure password authentication Then click Next The Congratulations window will open Internet Connection Wizard X Congratulations ou have successfully entered all af the information required to set up your account To save these settings click Finish 5 Confirm with Finish to complete the e mail account setup Setting up in Outlook 1 Open Outlook Express from the Start menu Start All programs Outlook Express 2 Select Tools and then Options The window below appears Rotor Gene Q User Manual 11 2012 7 107 Analysis User Interface Signatures Security Connection Maintenance General Read Receipts Send Compose General When starting go directly to my Inbox folder v Notify me if there are any new newsgroups M Automatically display folders with unread messages M Automatically log on to Windows Messenger Send Receve Messages M Play sound when new messages arrive Send and receive messages at startup Check for new messages every minute s IF my computer i nat connected at this time Do not connect Default Messaging Programs This application is the default Mail handler Make Default or This application is the default News handler Make Default Important To avoid any retrieval of e mails
73. Solution dNTPs and MgCl and RNase Free Water EpiTect MethyLight Master Mix for methylation specific real 59436 PCR Kit 200 time PCR analysis 200 x 50 ul reactions EpiTect HRM PCR Kit For 100 reactions 2x EpiTect HRM 59445 100 E 6 Master Mix containing HotStarTaq Plus DNA Polymerase EpiTect HRM PCR Buffer with EvaGreen dye dNTP Mix and RNase Free Water For up to date licensing information and product specific disclaimers see the respective QIAGEN kit handbook or user manual QIAGEN kit handbooks and user manuals are available at www qiagen com or can be requested from QIAGEN Technical Services or your local distributor Rotor Gene Q User Manual 11 2012 Appendix F Appendix F Liability clause QIAGEN shall be released from all obligations under its warranty in the event repairs or modifications are made by persons other than its own personnel except in cases where the Company has given its written consent to perform such repairs or modifications All materials replaced under this warranty will be warranted only for the duration of the original warranty period and in no case beyond the original expiration date of original warranty unless authorized in writing by an officer of the Company Read out devices interfacing devices and associated software will be warranted only for the period offered by the original manufacturer of these products Representations and warranties made by any person includ
74. WARNUNG Gefahr durch Stromschlag W21 ACHTUNG ffnen Sie den Rotor Gene Q nicht Durch das ffnen der Abdeckungen oder das Entfernen von Geh useteilen k nnen spannungsf hrende Komponenten freigelegt werden ACHTUNG Besch digung des Geh uses C8 Reinigen Sie das Geh use des Rotor Gene Q nie mit Alkohol oder alkoholhaltigen L sungen Alkohol sch digt das Geh use Verwenden Sie zum Reinigen des Geh uses ausschlie lich destilliertes Wasser Rotor Gene Q User Manual 11 2012 C 11 Appendix C 1 10 Symbole auf dem Rotor Gene Q Gef hrdung durch hei e Ger teteile in der Kammer k nnen Temperaturen von ber 120 C erreicht werden R ckseite des Ger ts Lesen Sie die Gebrauchshinweise im Handbuch C Typenschild auf der CE Kennzeichnung der EU Ger ter ckseite Konformit t Typenschild auf der Symbol der CSA Zertifizierung in Ger ter ckseite Kanada und den USA An der Rotorkammer bei ge ffnetem Deckel sichtbar Typenschild ae ac Hersteller i S d Gesetzes Ger ter ckseite WEEE Markierung Zertifizierung Typenschild auf der gem europ ischer Richtlinien Ger ter ckseite bzw Elektro und Elektronik Altger te Verordnung FCC Kennzeichen der Federal Communications Commission der Vereinigten Staaten C Tick Mark Zeichen f r Australien Herstellerkennung N17965 N 15128 Markierung gem RoHS Richtlinie f r China Beschr nkung der Verwendung bestimmter gef hr liche
75. ading is available in contrast to allelic discrimination which uses a cycle by cycle reading for each sample This means that the user must identify positive and negative controls to facilitate the analysis For the raw data signal levels are normalized relative to the known positive and negative controls for each channel The user then selects a percentage signal level as the threshold Terms used in EndPoint analysis Some terms used in EndPoint analysis are explained below Positive control This is a sample which is known to amplify Negative control This is a sample which is known not to amplify This represents the typical background signal Rotor Gene Q User Manual 11 2012 7 53 Analysis User Interface 7 54 Threshold Signal level Genotype The threshold is a signal level above which a sample is said to be positive amplified This setting must be adjusted by the user for each run percentage of fluorescent signal normalized so that the highest signal of the positive controls is 10096 and the lowest signal of the negative controls is 0 An interpretation of different permutations of reactions on different channels For example the genotype heterozygous could be assigned to samples that reacted in both channels green and yellow The genotype can also be used to report the results of reactions with internal controls For example results could be reported as inhibited positive or neg
76. al voltages inside the instrument When the instrument is connected to line power terminals may be live and opening covers or removing parts is likely to expose live parts To ensure satisfactory and safe operation of the Rotor Gene Q follow the advice below M The line power cord must be connected to a line power outlet that has a protective conductor earth ground Do not adjust or replace internal parts of the instrument Do not operate the instrument with any covers or parts removed If liquid has spilled inside the instrument switch off the instrument disconnect it from the power outlet and contact QIAGEN Technical Services If the instrument becomes electrically unsafe prevent other personnel from operating it and contact QIAGEN Technical Services the instrument may be electrically unsafe when m lt or the line power cord appears to be damaged m It has been stored under unfavorable conditions for a prolonged period m It has been subjected to severe transport stresses WARNING Electrical hazard W9 The instrument has an electrical compliance label which A indicates the voltage and frequency of the power supply as well as fuse ratings The equipment should only be operated under these conditions 1 4 Rotor Gene Q User Manual 11 2012 Safety Information 1 3 WARNING A CAUTION A 1 4 Environment Operating conditions Explosive atmosphere w10 The Rotor Gene Q is not designed for use in a
77. all the users of the computer are displayed Some accounts are used by the system and so will be unfamiliar The bottom pane shows the groups assigned to the user Rotor Gene Q User Manual 11 2012 7 91 Analysis User Interface BB User Admin Current User linrisangy Domain Groupe Selected Users Groups AG Analyst Administrators RG Operator gt Show Advanced Close 4 To assign a group to a user select the user s name from the list The bottom panel will update If the user has no groups they cannot launch the software In the example below we assign the user linsongy to the RG Analyst group by selecting the group on the left hand side then clicking the gt button Groups can be removed by selecting them then clicking the lt button 92 Rotor Gene Q User Manual 11 2012 Analysis User Interface BB User Admin x Current User linsanqu Users Administrator Guest labpc Groups Domain Groups Selected Users Groups Administrators AG Analyst RG Operator Show Advanced Close 5 Now log in as this user As an RG Analyst the Run menu and Profile button are unavailable However existing files can be opened and analyzed as shown in the screenshot below The status bar indicates that the user CORIT198 linsongy is an RG Analyst Rotor Gene Q User Manual 11 2012 93 Analysis User Interface E Rotor Gene Q Series Softwa
78. alysis User Interface standard reports see page 7 13 and in the Standard Curve window The slope is calculated as the change in divided by the change in log input e g copy number A 100 efficient amplification means a doubling of amplification product in each cycle resulting in an M value of 3 322 an amplification factor of 2 and a reaction efficiency of 1 Given an M value of 3 322 the calculations are as follows Exponential amplification 1061 3322 2 Reaction efficiency 10773322 1 1 As an alternative example an M value of 3 8 means that the reaction has an exponential amplification of approximately 1 83 and a reaction efficiency of 0 83 or 83 Offset In a formula describing the relationship between 2 variables the offset is expressed with the letter B y Mx B The offset is also sometimes referred to as the intercept B represents the for a given concentration of 1 unit By substituting 1 into the concentration formula as shown below C log 1 C 0 M B The result is C The intercept can change from run to run and is a less stable measurement than the gradient For this reason the gradient is analyzed more frequently than the intercept Main window The main window displays the amplification plots on a log scale Clicking Linear Scale at the bottom of the window changes the scale from the log scale to the linear scale and vice versa Changing
79. ame for the current selection The software recognizes these wells as replicates Rotor Gene Q User Manual 11 2012 79 Analysis User Interface Edit Samples ia d Jal x Fie Edt Format Security Standard Flotor Style 10 Name sample Standard The Rotor Style tab provides a cutdown version of the Standard tab and is designed for users who want to setup sample names and colors quickly It is not possible to define some settings such as the whether the sample represents a standard or the known concentration of each standard in this tab If these need to be defined the standard tab should be used Sample Page Suitability To access the Sample Page Suitability window click on More Options in the Edit Samples window and then click on Define Suitabilities The Sample Page Suitability window allows users to match sample pages to channels For example the sample page for the gene of interest may apply to the green channel and the sample page for the housekeeper gene may apply to the yellow channel In this example setting up sample page suitability reduces the number of analysis options available to just include those relevant for the particular assay 7 80 Rotor Gene Q User Manual 11 2012 Analysis User Interface The Sample Page Suitability window is shown below Sample Page Suitability X Sample Page Suitabilities enable ta
80. ample locking Locked templates Gain menu Window menu Help function 7 12 1 Send Support E Mail 8 1 8 2 8 3 8 4 8 5 8 6 10 10 1 10 2 10 3 11 11 2 Contents 4 Additional Functions Analysis templates Opening a second run Scaling options Exporting graphs Spanner wrench icon Selected area options Maintenance Procedures Optical Temperature Verification OTV principle Rotor Disc OTV Kit components Running an OTV High Resolution Melt Analysis Instrumentation Chemistry Rotor Gene Q User Manual 7 84 7 85 7 95 7 96 7 98 7 100 7 102 7 103 7 104 7 104 7 104 8 1 8 1 8 1 8 1 8 2 8 5 8 9 1 10 1 10 1 10 2 10 2 11 1 11 3 11 3 11 2012 Contents 11 3 SNP genotyping example 11 4 Methylation analysis example 11 5 Guidelines for successful HRM analysis 11 6 Sample preparation 11 7 Software setup 11 8 Real time PCR data analysis 11 9 HRM data analysis 12 Troubleshooting 12 1 Log Archives 12 2 HRM troubleshooting 12 3 General instrument errors 13 Glossary Appendix A Technical data Environmental conditions FCC Declaration Declaration of Conformity Waste Electrical and Electronic Equipment WEEE Appendix B Safety Information French FR 1 Informations de securite 1 1 Utilisation appropri e 1 2 S curit lectrique 1 3 Environnement Rotor Gene Q User Manual 11 2012 11 3 11 5 11 7 11 9 11 9 11 17 11 19 12 1 12 1 12 1 12 3 13
81. ample names are protected Integration with Windows security To provide a strong level of accountability Rotor Gene Q software does not manage security internally Accounts groups and passwords are all managed using the Windows built in security model Integration allows the same password that provides access to network files and programs to control Rotor Gene Q software access leading to less administration In larger organizations for example network administrators can easily remove access to ex users due to the centralized security model For this reason setting up the Rotor Gene Q software securely primarily involves configuration of the Windows security roles according to best practices Prerequisites To use security you must be running Windows XP Professional or Window 7 Professional The security features cannot be used with Windows XP Home or Windows 7 Home as they do not have the fine grained access model used by the software The software must be installed with the Force Authentication through Windows NT Logon option Note The Security menu will not appear if you are logged into a Linux Samba domain You must have either a local logon or a Windows server to use the security features 7 84 Rotor Gene Q User Manual 11 2012 Analysis User Interface 7 9 1 Configuration This section describes how to set up the system to run Rotor Gene Q software securely To use the security features the softwa
82. as Stat 554 End E28 Lin Elance 893 End Confidence Pucana Fionaan Sere Soles 1 7 Buel 54 Rotor Gene Q User Manual 11 2012 High Resolution Melt Analysis 6 Genotypes will be called automatically by the software in the Results window A confidence value is provided as an integrity check of autocalled results The threshold value above which autocalls are made can be edited Samples that fall below the set threshold will be marked as a variation for closer investigation or retesting HRM Results Melt A from 82 FarRed Page 1 Confidence mutation heterozygote mutation heterozygote heterozygote wild type wild type heterozygote heterozygote wild type wild type Normalisation Regions Leading Range 24 Start 182 4 I End 828 10 x Trailing Range Start 189 3 End 189 7 Confidence Percentage Threshold 30 z Rotor Gene Q User Manual 11 2012 11 23 High Resolution Melt Analysis This page intentionally left blank 11 24 Rotor Gene Q User Manual 11 2012 Troubleshooting 12 Troubleshooting 12 1 Log Archives The software keeps an unmodified record of each run along with diagnostic information in its Log Archive repository By using the Help Send Support Email option you can send an e mail along with all the necessary diagnostic information to QIAGEN Technical Service see Section 7 12 1
83. assword 5 M User must change password at next logon User cannot change password Password never expires Account is disabled 6 Click Create You can now log on as this user Assigning roles to each user You should now assign roles to each user Access is divided into the following areas M Rotor Gene Q Operator can perform runs but cannot generate reports or perform analysis Rotor Gene Q Analyst can analyze run data and generate reports but cannot perform new runs Rotor Gene Q Operator and Analyst has the capabilities of both roles Administrator can unlock sample names and perform all operations of Analysts and Operators None access to the software is denied 7 90 Rotor Gene Q User Manual 11 2012 Analysis User Interface To assign roles 1 Log in to Windows as an administrator or use the Rotor Gene Q Software Login icon to open the software and log in Run Rotor Gene As Other User Domain coRIT1 38 OF Username linongs Cancel Password o 2 Once the software is open click on the Security menu The first time the Security menu is accessed Rotor Gene Q software configures a number of system groups that will control access to the software Rotor Gene 0 Series Software X 3 R Groups do nat exist an the domain Would vau like them to be created De 3 Click Yes The User Admin window appears In the top panel
84. at are hot WARNING Risk of personal injury and material damage 6 CAUTION Ifthe equipment is used a manner not specified by the manufacturer the protection provided by the equipment may be impaired WARNING Risk of personal injury and material damage W7 CAUTION Loose paper underneath the Rotor Gene Q interferes with instrument cooling It is recommended that the area beneath the instrument is kept free of clutter CAUTION Damage to the instrument C2 Always use a locking ring on the rotor This stops caps from coming off tubes during an experiment If caps come off during an experiment they may damage the chamber CAUTION Damage to the instrument C3 Visually inspect and make sure the rotor is not damaged or deformed before each run IF you touch the Rotor Gene Q during an experiment while you are charged with static electricity in severe cases the Rotor Gene Q may reset However the software will restart the Rotor Gene Q and continue the experiment Rotor Gene Q User Manual 11 2012 1 3 Safety Information 1 2 Electrical safety Disconnect the line power cord from the power outlet before servicing WARNING Electrical hazard w8 Any interruption of the protective conductor earth ground lead inside or outside the instrument or disconnection of the protective conductor terminal is likely to make the instrument dangerous Intentional interruption is prohibited Leth
85. at the users of this product must be informed of the following facts and circumstances This device complies with part 15 of the FCC Operation is subject to the following two conditions 1 This device may not cause harmful interference and 2 this device must accept any interference received including interference that may cause undesired operation This Class B digital apparatus complies with Canadian ICES 0003 The following statement applies to the products covered in this manual unless otherwise specified herein The statement for other products will appear in the accompanying documentation Note This equipment has been tested and found to comply with the limits for a Class B digital device pursuant to Part 15 of the FCC Rules and meets all requirements of the Canadian Interference Causing Equipment Standard ICES 003 for digital apparatus These limits are designed to provide reasonable protection against harmful interference in a residential installation This equipment generates uses and can radiate radio frequency energy and if not installed and used in accordance with the instructions may cause harmful interference to radio communications However there is no guarantee that the interference will not occur in a particular installation If this equipment does cause harmful interference to radio or television reception which can be determined by turning the equipment off and on the user is encouraged to try
86. ated correctly during sample setup see Section 6 1 4 In particular corresponding samples must have the same name in each analysis In a multiplex reaction where the tube positions of the gene of interest and the normalizing gene are the same one set of sample definitions is sufficient If performing relative analysis with a normalizing gene using a single channel i e reactions are run in separate tubes Rotor Gene Q User Manual 11 2012 7 31 Analysis User Interface using the same fluorophore then 2 sample pages should be created The first should label the tube positions with sample names for the gene of interest with the other positions left unnamed The second should label the positions used for the normalizing gene The software will then match samples across the 2 analyses based on their names Expression analysis using the two standard curve method Data can first be analyzed for each gene using quantitation analysis Otherwise the results for each gene will automatically be determined using the Autofind Threshold tool 1 From the Analysis window select the 2 Std Curve Rel tab Click New Analysis Quantitation Melt 2 Std Curves Rel Other Analysis Show Hide M Auto shrink window 2 Enter a name for the analysis 7 32 Rotor Gene Q User Manual 11 2012 Analysis User Interface Rotor Gene Q Series Software l x Enter a name for the relative quantita
87. ative depending on whether a reaction was seen in certain channels or not Rotor Gene Q User Manual 11 2012 Analysis User Interface Profile configuration x 1 Rotor Selection 2 Confirm Profile H 3 New Open Save As Help The run will take approximately 6 minute s to complete The graph below represents the run to be performed Click on a cycle below to modify it Hold Insert after Insert before Remove This cycle repeats 5 time s Click on one of the steps below to modify it or press or to add and remove steps for this cycle Timed Step 50 deg 10 seconds Acquiring to Cycling amp on Green Long Range Touchdown 50 deg for 10 secs lt Back Save Template Start Run To perform EndPoint analysis perform a profile with a hold at 50 C for several minutes then a cycling step with 1 step 50 C for 10 seconds acquiring on the required channel Set the number of repeats to 5 as shown above These times are a guide only and may vary for your particular application The more repeats in the profile the more information available to perform the analysis The analysis will automatically average all the readings to achieve a single value for each sample There is no specific number of repeats required Unless a very high level of accuracy is required 5 repeats are usually sufficient Analysis EndPoint analysis can be performed on a number of channels si
88. ative controls are analyzed and the one with the lowest fluorescence level is set to be O96 Rotor Gene Q User Manual 11 2012 7 57 Analysis User Interface 7 58 3 The raw fluorescence values of the remaining samples are scaled relative to the highest positive control and the lowest negative control For example Type Fluorescence Positive control 56 3 Positive control 53 0 Negative control 4 5 Negative control 4 3 Sample 48 1 Sample 6 4 This run was a success as the 2 positive and 2 negative controls are close together and are outside the fluorescence values of the samples The normalized values are Type Expression Positive control 100 0 Positive control 93 7 Negative control 0 4 Negative control 0 0 Sample 84 2 Sample 4 0 Sample 1 was the positive control with the highest fluorescence so it was set to 100 The other positive control was slightly lower Sample 4 the lowest negative control was set to 0 It is now clear that sample 5 has probably amplified whereas sample 6 has probably not amplified Note Depending on the positive and negative controls selected it is possible to achieve expression levels of greater than 100 or less than 0 A result of greater than 100 can be interpreted to mean that the sample is more highly Rotor Gene Q User Manual 11 2012 Analysis User Interface expressed than the positive controls A result of less than 0 can be interpreted to mean tha
89. c cet appareil peuvent tre dangereux ou le devenir apr s l ex cution du cycle du protocole Portez toujours des lunettes de protection des gants et une blouse de laboratoire La personne responsable par exemple le directeur du laboratoire doit prendre les pr cautions n cessaires afin de garantir que l environnement de travail est s r et que les op rateurs de l instrument ne sont pas expos s des niveaux dangereux de substances chimiques ou biologiques toxiques comme cela est d fini dans les fiches techniques sant s curit MSDS ou dans les documents de l OSHA de l ACGIH ou du COSHH applicables L evacuation des vapeurs et la mise au rebut des d chets doivent s effectuer conform ment toutes les r gles et lois nationales r gionales et locales relatives la sant et la s curit OSHA Occupational Safety and Health Administration tats Unis d Am rique administration pour la sant et la s curit du travail t ACGIH American Conference of Government Industrial Hygienists tats Unis d Am rique Conf rence am ricaine des hygi nistes industriels gouvernementaux COSHH Control of Substances Hazardous to Health Royaume Uni contr le des substances dangereuses pour la sant Vapeurs toxiques Si vous travaillez avec des solvants volatils ou des substances toxiques vous devez disposer d un syst me de ventilation de laboratoire efficace afin d vacuer les vapeurs qui
90. cation and are not due to the Rotor Gene Q Similar tests performed on block based cyclers would yield greater variation due to the lower temperature uniformity of block based systems To compare cyclers if desired we recommend comparing the standard deviation of the C value Note More detailed information on confidence intervals is available in Appendix D Note Each of the columns can be displayed or hidden by right clicking on the window and then selecting or deselecting the column name Rotor Gene Q User Manual 11 2012 7 23 Analysis User Interface Quant Results Cycling A Green Page 1 _ nml x Type Ct Ej pales 1 10e8 Standard nm UU 2 10e8 Standard w No 3 10e8 Standard Colour 4 10e Standard NEG w Name 5 10e7 Standard HEC w Type 10 Standard w Ct ri 10e6 Standard 11 46 w Ct Comment 10e6 Standard 11 39 w Given Conc copies reaction 3 10e6 Standard 11 40 w Calc Conc copies reaction 10 10 5 Standard 15 04 w Var 11 1065 Standard 15 03 v Rep Ct 12 1065 Standard 75 05 v Rep Ct Std Dev 13 10 4 Standard 19 26 v Rep Ct 95 CI 14 10e4 Standard 16 45 An cuit EDI 15 10 4 Standard 18 23 16 10e3 Standsd 2225 USE 17 10e3 Standard 22 17 1 00E 03 1 02E 03 18 10e3 Standard 22 31 1 00E 03 9 41E 02 13 We Standard 26 01 1 00 02 8 92E 01 0 10 2 Standard 25 72 1 00 02 1 0 E 02 21 Hee Standard 25 94 1 00 02 9 33E 0
91. ccess the reaction chamber The following items are already installed inside the Rotor Gene M 72 Well Rotor this rotor is blue in color 72 Well Rotor Locking Ring A laptop computer may be included in the packaging depending on your order details Accessories Rotor Discs and accessories can be ordered separately for use with the Rotor Gene Q For details see Appendix E Hardware installation Once the Rotor Gene Q has been unpacked proceed with installation as described below Damage to the instrument C7 When Rotor Gene Q is started immediately after delivery in cold climates mechanical parts can block Allow the instrument to acclimatize to room temperature for at least an hour before turning the instrument on Rotor Gene Q User Manual 11 2012 Installation Procedures 1 Place the Rotor Gene Q on a level and vibration free surface 2 Ensure that there is sufficient space behind the instrument for the lid to open fully 3 Ensure that the power switch at the back of the instrument can be reached easily 4 Do not obstruct the back of the instrument Ensure that the power cord can be easily detached if required to disconnect power to the instrument 5 The Rotor Gene Q software should be installed before the laptop computer is connected to the Rotor Gene Q Please refer to Section 4 7 below or the Rotor Gene Q Installation Guide provided with the instrument on how to install the Rotor Gene Q
92. ced or specialized uses of our products This information is helpful to other scientists as well as to the researchers at QIAGEN We therefore encourage you to contact us if you have any suggestions about product performance or new applications and techniques For technical assistance and more information call one of the QIAGEN Technical Services Departments or local distributors see back cover For up to date information about the Rotor Gene Q visit www qgiagen com RotorGeneQ 2 2 2 Policy statement It is the policy of QIAGEN to improve products as new techniques and components become available QIAGEN reserves the right to change specifications at any time In an effort to produce useful and appropriate documentation we appreciate your comments on this user manual Please contact QIAGEN Technical Services 2 2 3 Version management This document is the Rotor Gene Q User Manual version 3 for Rotor Gene Q instruments using Rotor Gene Q software versions 2 1 0 or higher 22 Rotor Gene Q User Manual 11 2012 Introduction 2 3 Intended use of the Rotor Gene Q The Rotor Gene Q instrument is designed to perform real time and end point thermal cycling using the polymerase chain reaction PCR and high resolution melting analysis HRM in molecular biology applications as well as for other applications such as concentration measurement protein analysis and enzyme kinetics The Rotor Gene Q if used in combinati
93. centage is relative to the largest maximum change found in any tube For example if one sample began at a background of 2 Fl and increased to 47 FI then 45 Fl represents 10096 An NTC Threshold of 1096 would consider any sample less than 4 5 Fl as noise The Reaction Efficiency Threshold is an alternative method of excluding noise from analysis This normalizing algorithm uses the reaction efficiency estimation techniques used in comparative quantitation see Section 7 6 6 All samples that do not have a reaction efficiency of at least this level are excluded and a flag NEG R Eff will be shown in the CT Comment column A level of 096 indicates that during the exponential phase no reaction took place 10096 indicates that a completely efficient reaction took place during the exponential phase Negative percentages indicate that during the exponential phase the fluorescent signal declined Current research is not conclusive on the precise levels of efficiency needed to distinguish genuine reactions from contamination and other effects For this reason we recommend using this feature conservatively with the assumption that any sample with a genuine reaction will have some visible exponential phase with 7 28 Rotor Gene Q User Manual 11 2012 Analysis User Interface some increase in fluorescence Setting this value higher than 0 will exclude some samples with inefficient but perceptible increase in fluore
94. cles fi Before E Eliminate Cycles To set click on the icon a grid with a red before arrow then click and hold on the graph and drag the line to the right This eliminates the threshold for low cycle numbers Note This is useful when there is noise during the initial cycles for example due to sample mixing effects Rotor Gene Q User Manual 11 2012 7 19 Analysis User Interface Auto Find This function scans the selected region of Threshold the graph to find a threshold setting which delivers optimal estimates of given concentrations The selected region can be changed by entering new upper and lower bounds in the text boxes which appear For most analyses the default upper and lower bounds are suitable The range of threshold levels is scanned to obtain the best fit of the standard curve based on the samples that have been defined as standards i e where the R value is closest to 1 0 Threshokd Results This opens the Quantitation Results window By default this window is opened when an analysis is opened If it has been closed it can be reopened using this command Quant Resets Cycling AGreen Page 1 E Cycling Green Page 1 Gieen Page 1 2 Cycling A Gisan P 1 Cycling Green Page 1 7 LI DE 07 07 ox F 005 5 91 68 21 LA 07 3 29 Of 673 07 Cycling A Gisan Page 1 Cycling Green Page 1 Cycing A Green Page 1 7 b Stand 3 UE
95. d 4 Click the Reports button to display the Report Browser Select the analysis with the correct name from the list Click the Show button to display the relative quantitation report The Export option exports the results to a new Excel spreadsheet If a calibrator is 7 34 Rotor Gene Q User Manual 11 2012 Analysis User Interface included the results are calculated relative to the calibrator sample which is assigned a value of 1 loxi Report Categories Templates General amp Melt Curve Analysis Comparative Quantitation e Delta Delta CT Relative Quantitation Report 2 Standard Curves Full Report Report 2 Standard Curves Relative Quant Analysis E B 2 Standard Curves Relative Quantitation s Relative Quant Standard Curve Relative Quant Analysis The concentrations as read from the standard curves for the gene of interest GOI Conc and the normalizing gene Norm Conc as well as the relative concentration Relative Conc are displayed The results can be saved as a Word file ae EEE d Exprestin E grs Leggi i Rotor Gene Q User Manual 11 2012 7 35 Analysis User Interface Relative Quant Analysis Experiment Information Gene Expression Graph Raw Values i j Gene Expression Graph Logarithmic I A 6 The Rel Min and R
96. d Groups can be used to perform advanced user management tasks Secure Boot Settings T IE is recommended that vou require users to press CEri AlE Delete before logging en This ensures password security and helps protect the system From harmful programs M Require users to press Ctrl Alt Delete before logging on 7 88 Rotor Gene Q User Manual 11 2012 Analysis User Interface 4 In the window that appears select the Users folder Right click on the right hand window and select New User oix Action view lt ABA 3 administrator Built in account Ij analyst Rotor Gene analyst Guest Built in account 115 NEWLA Internet Guest Account Built in account Internet Guest Account Built in account B matthew Matthew Herrmann IB operator Rotor Gene Operator Wi testuser Rotor Gene Analyst and Ij vUSR_NEWL 4 VSA Server Account Account For the New User Creates a new Local User account Refresh ooo e Export list View b Arrange Icons gt Line up Icons Help 5 Enter a username and password By default the user will be created with normal access privileges This means they can run software but not install new programs or change system settings Rotor Gene Q User Manual 11 2012 89 Analysis User Interface LIAE 2x User name 000 00000000 Full name New User 00 0000000 Description Password E Contirm p
97. d alimentation situ l arri re de l appareil et d branchez le c ble d alimentation de la prise de courant B 2 Rotor Gene Q User Manual 11 2012 Appendix B Note Ne pas d brancher l instrument alors qu il est en marche mis part pour les raisons indiqu es dans ce manuel Une coupure de courant pendant la marche a des effets incalculables sur les chantillons et les r sultats de l analyse Il est votre propre risque de continuer un run apr s avoir d branch l instrument AVERTISSEMENT Risque d accident corporel et de d t rioration ATTENTION du mat riel 4 N essayez pas d ouvrir le couvercle en cours d exp rience ou pendant que le Rotor Gene tourne Si vous ouvrez le verrou du couvercle et mettez les mains l int rieur de l appareil vous risquez de toucher des pi ces chaudes aliment es en lectricit ou tournant grande vitesse Vous pouvez alors vous blesser et endommager l appareil AVERTISSEMENT Risque d accident corporel et de d t rioration ATTENTION du mat riel 5 Si vous devez arr ter un essai rapidement teignez l appareil puis ouvrez le couvercle Laissez refroidir la chambre avant de mettre les mains l int rieur de l appareil Sinon vous risquez de vous blesser en touchant des pi ces chaudes AVERTISSEMENT Risque d accident corporel et de d t rioration ATTENTION du mat riel we Toute utilisation de l quipement non conforme aux sp cifications d
98. d click To remove all acquisitions click lt lt Chart gt gt Dont Acquire Help Dye Channel Selection Chart FAM SybrGreen alexad88 4 JOE CalGold Cal range TET Yakima Yellow VIC Crimson Quasar705 LCRed705 80 8 Set HRM run conditions Modify the conditions to suit the amplicon For the first set of experiments allow for a wide melt domain Use the theoretical T as a guide to a suitable range Once you have determined where the product will melt reduce the melt domain to no greater than 10 C Ensure that the start of the melt occurs 5 C prior to the first melt transition The default ramp is set to 0 1 C with a hold of 2 seconds at each step The minimum ramp transition is 0 05 C with a second hold at each step Data is automatically acquired to the HRM channel Automatic Gain Optimisation is performed by default The software will search for the optimal gain setting so that the highest fluorescence value reported is no greater than 7O units on a scale of 100 Note that this can be increased to a maximum of 100 11 14 Rotor Gene Q User Manual 11 2012 High Resolution Melt Analysis x 1 amp 3 New Open Save As Help The run will take approximately 104 minute s to complete The graph below represents the run to be performed Click cycle below to modify it Hold Insert after Cyclin Insert before Remove Ramp from 70 d
99. dentifying these outliers and filtering them out of subsequent HRM analysis will greatly improve the overall effectiveness of HRM analysis since analyzing poor quality PCR product will result in poor HRM results We recommend analyzing quantitative real time PCR data as follows 1 Analyze the real time data using the Quantitation option from the Analysis window If any C values are 30 or higher the corresponding reactions are considered to have amplified too late These samples must be analyzed with suspicion or removed from the analysis as an outlier Late amplification is usually due to too little starting template amount and or high levels of sample degradation Rotor Gene Q User Manual 11 2012 11 17 High Resolution Melt Analysis 2 Assess the end point fluorescence level If end point fluorescence in any of the amplification plots is low compared with the majority of plots in the data set omit those samples from the analysis even if their value is less than 30 Low end point fluorescence can indicate incorrect dye amount incorrect levels of reaction components such as primers or the action of inhibitors 3 Use the Comparative Quantitation option from the Analysis window to obtain the reaction efficiency of each sample If the efficiency is not similar to other reactions in the experiment or is less than approximately 1 4 omit the reaction as an outlier Comparative Quantitation Analysis Cycl
100. during PCR runs disable the default entries in the Send Receive Messages screen 3 Disable Send and receive messages at startup 4 Disable Check for new messages every 30 minutes 5 Confirm changes with 7 108 Rotor Gene Q User Manual 11 2012 Additional Functions 8 2 8 3 Additional Functions Analysis templates Some analyses require the user to define thresholds normalization settings and genotype settings Often these settings are reused frequently in multiple experiments Analysis templates enable the user to save and reuse these settings This reduces the effort of reentering settings and reduces the risk of error Quantitation Melt Allelic discrimination Scatter graph analysis and EndPoint analysis support analysis templates These analyses allow the user to export a template that is unique to the analysis e g Quantitation analysis allows export and import of qut files that contain quantitation settings After an analysis template has been imported or exported the filename of the template is displayed for future reference Imported Settings lt lt 1 emplates EC qut Import Export Opening a second run While performing a run it is possible to open and analyze runs that were performed earlier Several functions such as New or Start Run buttons are not activated in the second window A new run can be started from the first window onc
101. e Delete the file C Program Files Rotor Gene Q Software Templates normal ret and then restart the software This error occurs when the software has attempted to set the rotor speed and failed to set the target speed within a time out period Further investigations are required by a QIAGEN Field Service Specialist to diagnose the problem with the instrument Please contact your distributor or QIAGEN Technical Service This error occurs when the software tries to connect to the machine on the configured COM port when the port is being used by another software Close any applications such as communications or synchronization software and then retry This error occurs when the software has issued shutdown command to shut down the instrument and the machine keeps sending data back after an expected grace period of time Reset the machine and restart the software 12 6 Rotor Gene Q User Manual 11 2012 Troubleshooting Error message Temperature Protection Activated The instrument detected that the chamber temperature increased above a safe level It has therefore entered a self protection mode Please turn off the instrument and contact your distributor if the problem persists Thermistor Is Open The instrument detected that the thermistor is open and so to prevent damage to the machine it has been turned off Please contact your distributor if this occurs again Unrecoverable errors occurred
102. e the f rst run has finished Scaling options To access Adjust Scale click on Adjust Scale at the bottom of the main window or right click on the graph and select Adjust Scale on the menu that appears A scale can be manually entered in the window that appears Rotor Gene Q User Manual 11 2012 8 1 Additional Functions Adjust Scale Maximum Minimum aj To access Auto Scale click on Auto Scale at the bottom of the main window or right click on the graph and select Auto Scale on the menu that appears Auto Scale attempts to fit the scale to the maximum and minimum readings in the data To access Default Scale click on Default Scale at the bottom of the main window or right click on the graph and select Default Scale on the menu that appears Default Scale resets the scale to display from O to100 fluorescence units 8 4 Exporting graphs Picture export The following steps describe how to save an image 1 Right click on the image and select Export from the menu that appears 2 The Export Dialog window appears Select the desired format from the Format list 8 2 Rotor Gene Q User Manual 11 2012 Additional Functions Export Dialog ES Picture Native Data Options as Metatile oo rx as PostScript Monochrome 3 Select the Size tab and specify the desired size Export Dialog Optio
103. e are averaged to produce the amplification value that is shown on the right of the screen under the toggler The greater the variation between the estimated amplification values of each sample the larger the confidence interval will be indicated by the value after the sign The confidence interval for a large sample number N gives a 68 3 probability that the true amplification of the samples lies within this range 1 standard deviation By doubling the interval a 95 4 confidence interval for a large N is achieved Calibrator Replicate As in the delta delta C method a calibrator sample is required and measurements are relative to this calibrator sample Replicates of the calibrator can be analyzed since if multiple sample positions have the same name the average of the takeoff points of these samples will be used To use this feature correctly ensure that replicates have identical names Calibrator Replicate Results Average Amplification 1 78 0 04 The average amplification is used to calculate expression For example a sample with a low amplification value will take longer to reach a certain absolute copy number than a sample with a higher amplification value The Rep Conc column of the Comparative Quantitation Results window provides the relative concentration The relative concentration of each sample compared with the calibrator sample is calculated based on the takeoff point and the reaction efficienc
104. e considered to be genotyping samples Click on the icon to the right of each text box then drag the threshold on the graph to set these values visually Discrimination Threshold Theodor 7 48 Rotor Gene Q User Manual 11 2012 Analysis User Interface Genotypes This opens the Genotyping window which is used to define which genotype is detected in each channel This window enables genotypes to be assigned to channels for allelic discrimination analysis In the example below a sample is heterozygous if readings in channels Cycling A Green and Cycling A Yellow cross the threshold 8 Genotyping Reacting Channels Wild Type Cycling Green Heterozygous Cycling Green Cycling A yellow Mutant Cycling A rellaw Cancel Help Allelic analysis Allelic analysis templates enable export of templates normalization threshold and genotype settings into a single alt file This file can be imported and reapplied in other experiments See Section 8 1 for more details Imported Settings Import Export 7 6 8 Scatter graph analysis Scatter graph analysis enables genotyping based on the relative expression of amplification plots across 2 channels Unlike allelic discrimination genotype is decided based on regions defined from the scatter graph rather than from a Rotor Gene Q User Manual 11 2012 7 49 Analysis User Interface single threshold To perform this analysis
105. e from the list under the Advanced tab of the New Run window Rotor Gene Q User Manual 11 2012 6 7 Operating Procedures Software x Quick Start Advanced Imports the cyding and acquisition and sample definitions T from the last run open silos in the software erform Last Run LL Three Step with Melt Two Step Other Runs t Instrument Maintenance is m Locked Templates Open A Template In Another Folder New Cancel Help M Show This Screen when Software Opens Template options provided in this window are similar to those provided when using the Quick Start wizard Section 6 1 Perform Last Run Perform Last Run imports the cycling acquisition and sample definitions from the last run open in the software Empty Run This is an empty run which allows the user to define all parameters of the profile Three Step with This is a three step cycling profile and a Melt melt curve with data acquisition on the green channel 6 8 Rotor Gene Q User Manual 11 2012 Operating Procedures Software 6 2 1 Two Step HRM Other Runs Instrument Maintenance This is a two step cycling profile with data acquisition on the green channel only to speed up the run This folder contains 2 high resolution melt profiles This folder contains additional profiles This contains t
106. e instrument housing with alcohol or A alcohol based solutions Alcohol will damage the housing To clean the housing use distilled water only Rotor Gene Q User Manual 11 2012 1 9 Safety Information 1 10 Symbols on the Rotor Gene Q Symbol Location Description Heat hazard the temperature of the chamber can reach temperatures above 120 C 248 F ce 17 plate on the back CE marking for European of the instrument Conformity Type plate on the back CSA listing mark for Canada of the instrument and the USA Type plate on the back Legal manufacturer of the instrument Type plate on the back Waste Electrical and Electronic of the instrument Equipment WEEE FCC mark of the United States Type plate on the back Federal Communications of the instrument Commission Type plate on the back C Tick mark for Australia of the instrument supplier identification N17965 Near the sample chamber visible when lid is open RoHS mark for China the Type plate on the back restriction of the use of certain hazardous substances in electrical and electronic equipment of the instrument 1 10 Rotor Gene Q User Manual 11 2012 Introduction 2 Introduction Thank you for choosing the Rotor Gene Q We are confident it will become an integral part of your laboratory Before using the Rotor Gene Q it is essential that you read this user manual carefully and pay particular attention to t
107. e le calibrage des fusibles L appareil ne doit tre mis en marche que sous certaines conditions Rotor Gene Q User Manual 11 2012 B 5 Appendix B 1 3 Environnement Conditions de fonctionnement AVERTISSEMENT Atmosph re explosive W10 Le Rotor Gene Q n est pas concu pour tre utilis dans une atmosph re explosive ATTENTION D t rioration de l appareil C4 1 4 A La lumiere directe du soleil peut decolorer des pieces de l appareil et endommager les pi ces en plastique Le Rotor Gene Q doit tre plac l abri de la lumi re directe du soleil S curit biologique Les sp cimens et les r actifs contenant des mati res provenant de sources biologiques doivent tre consid r s comme potentiellement infectieux Utilisez des proc dures de laboratoire s res comme d crit dans des publications telles que Biosafety in Microbiological and Biomedical Laboratories HHS www cdc gov od ohs biosfty biosfty htm Echantillons Les chantillons peuvent contenir des agents infectieux Vous devez conna tre le risque que de tels agents repr sentent pour la sant et devez utiliser stocker et mettre au rebut ces chantillons conform ment aux r gles de s curit n cessaires B 6 Rotor Gene Q User Manual 11 2012 Appendix B AVERTISSEMENT Echantillons contenant des agents infectieux wi Certains chantillons utilis s avec cet appareil peuvent contenir des agents infectieux Manipu
108. e list then click Select This window will close and you will be taken to the main window of this analysis E Green Cycling Arange af Gene of Interest Select Other Run Cancel 5 Click the Reports button to display the Report Browser Select the analysis with the correct name from the list Click the Show button to display the relative quantitation report The Export option exports the results to a new Excel spreadsheet If a calibrator is included the results are relative to the calibrator sample which has a value of 1 E Report Browser Report Categories Templates Jnl x General Melt Curve Analysis d Comparative Quantitation Delta Delta CT Relative Quantitation o Report ipea Delta CL Fall Report MIQE Report Delta Delta Ct Condse Relative Quant Analysis T A h anti 8 2 Standard Curves Relative Quantitation s Relative Quant Delta Delta Relative Quant Analysis An example of results from this analysis is shown below The C values for the gene of interest GOI CT C values for the normalizing gene Norm CT Delta C Delta Delta Delta C and relative concentration Relative Conc are displayed The expression is relative Rotor Gene Q User Manual 11 2012 7 39 Analysis User Interface to the calibrator sample which is assigned a relative expression of 1 For further informatio
109. e quantitation analysis settings must have been saved in the run file This lists the analyzed channels and their standard curve formulas In this area M and B values can be entered directly This is useful in cases where the values are from an external source such as an Excel spreadsheet Some chemistries produce a fluorescent signal that decreases exponentially instead of increasing It is possible to analyze these data using Quantitation but the Invert Raw Data checkbox should be checked For all other quantitation analysis this option should remain unchecked Invert Raw D ata Rotor Gene Q User Manual 11 2012 Analysis User Interface Cr Calculation The C value is the cycle number at the point where the amplification curve crosses a threshold of detection By setting a threshold line and calculating the intersection with each of the curves the value for each sample is established Threshold To set the threshold click on the icon a grid with a red arrow then click and hold on the graph and drag the line to the desired level Alternatively enter a log value Alternatively Auto Find Threshold can be used to automatically determine the threshold When setting a threshold manually it should be set in the exponential phase of the run significantly above the background level to avoid noise and below the onset of signal plateau in later cycles CT Calculation Threshold 0 0353 Eliminate Cy
110. ed Rotor Gene Q User Manual 11 2012 4 1 Installation Procedures 4 2 4 3 4 2 AC Power connection Power requirements The Rotor Gene Q operates at M 100 240 V AC 50 60 Hz 560 VA peak Make sure that the voltage rating of the Rotor Gene Q is compatible with the AC voltage available at the installation site Mains supply voltage fluctuations are not to exceed 10 of nominal supply voltages Grounding requirements To protect operating personnel QIAGEN recommends that the Rotor Gene Q be correctly grounded earthed The instrument is equipped with a 3 conductor AC power cord that when connected to an appropriate AC power outlet grounds earths the instrument To preserve this protection feature do not operate the instrument from an AC power outlet that has no ground earth connection Installation of AC power cord Connect the suitable end of the AC power cord to the socket located at the rear of the Rotor Gene Q instrument and the other end to the AC power outlet PC requirements The laptop computer optionally supplied with the Rotor Gene fulfills the requirements of the Rotor Gene software detailed in the following table Rotor Gene Q User Manual 11 2012 Installation Procedures PC system requirements Description Minimum requirement Operating system Microsoft Windows XP Professional edition 32 bit Microsoft Windows 7 Professional edition 32 bit Processor Intel C
111. egrees to 33 degrees Rising by 0 1 degree s each step Wait for 90 seconds of pre melt conditioning on first step ait for 2 seconds for each step afterwards Acquire to 7 on HRM Gain Optimisation v Optimise gain before melt on all tubes The gain giving the highest fluorescence less than 70 will be selected 9 Optional Set Auto Gain Optimisation This applies to the real time amplification step only and is set for the green channel Click the Optimize Acquiring button to optimize only those channels used by a run Optimization is best performed just before the first acquisition step so check the Perform Optimization Before First Acquisition checkbox The recommended background fluorescence range for intercalating dyes is between 1 and 3 Fluorescence units To change this setting click the channel name to select it in the list and then click the Edit button Rotor Gene Q User Manual 11 2012 11 15 High Resolution Melt Analysis Auto Gain Optimisation Setup Optimisation Auta G ain Optimization wall read the Fluaresence on the inserted sample at S en different gain levels until it finds one at which the fluorescence levels are y acceptable The range of fluorescence you are looking for depends on the chemistry vau are performing Set temperature to EU degrees Optimize All Optimise Acquiring v Perform Optimisation Before 1st Acquisition Perform Opt
112. el Max values are generated by calculating the standard deviation of the quotient from the standard deviations of the GOI and Normalizer using the following formula 2 2 C Y on C Vaoi E CV Norm where S stddev CV X meanvalue 7 6 4 Delta delta C relative quantitation The delta delta C method enables relative gene expression analysis It is described by Livak and Schmittgen 2001 This method does not require standard curves to be included in each run Each sample is first normalized for the amount of template added by comparison with the normalizing gene Livak K J and Schmittgen T D 2001 Analysis of relative gene expression data using real time quantitative PCR and the 2 delta delta C T method Methods 25 402 7 36 Rotor Gene Q User Manual 11 2012 Analysis User Interface These normalized values are further normalized relative to a calibrator treatment The calibrator could be for example wild type untreated control or time zero samples It is essential that the amplification efficiencies of the gene of interest and the normalizing gene are identical and that this is validated according to the guidelines of Livak and Schmittgen It is essential that the sample names are defined correctly in the Edit Samples window with the same samples labeled identically in each composite quantitation analysis 1 Analyze the data using Quantitation It is not necessary to run
113. employs a background task M Any software that checks for updates on the internet E Remote monitoring and management tools Please be aware that due to the dynamic nature of the IT world this list may not be complete and tools may be released that are not known at the time of writing It is important that system administrators take care that such a tool is not active during a PCR run Rotor Gene Q User Manual 11 2012 4 11 Installation Procedures 4 9 3 4 10 Operating system updates We strongly recommend turning off any automatic update processes of the operating system on the computer that is used for data acquisition on the Rotor Gene Q We are closely monitoring available updates and will notify customers whenever an update is considered to be safe and important to be installed Updating software Software updates are available from the QIAGEN Web site at www qiagen com RotorGeneQ Online registration is necessary to download the software Rotor Gene Q User Manual 11 2012 Operating Procedures Hardware 5 Operating Procedures Hardware This section describes operation of the Rotor Gene Q 5 1 Rotor types First select which tube type and rotor to use There are 4 rotors available to accommodate different tube types Note 36 Well Rotor and 72 Well Rotor are delivered with the instrument The Rotor Disc Rotors are accessories IMPORTANT Use identical tubes in a run Do not mix different tube
114. ence End 87 4 Confidence Pescontage Threshold 0 x Rete Gene Q Senes Software 1 7 Bukid 94 4 Click on the Genotypes button to define the genotypes Input each genotype category name and select a representative sample for each from the sample list LIII mx mutation wild type heterozygote OK Cancel d Rotor Gene Q User Manual 11 2012 11 21 High Resolution Melt Analysis 11 22 y Select a Control X Please select the control that is representative of this genotype Currently Selected 198 21197 3 negl 41196 5 195 6 2 7 202 8 201 ra ann Select Select None Cancel 5 View the difference plot by selecting the Difference Graph tab Then select the genotype you wish to compare all other samples against using the drop down menu at the top of the window In the example shown all samples are plotted subtracted from an average plot of all samples labeled Mutation 1 Rober Gene Q Series Soltware high resolution melt demo EM hormalised Graph Mel fron B7 Farked Page ala a Analyse Hun View Windle ds i LER PUES SD z amp Hew One awe Get Py Son Sellin Progen Profle Temp Samples Reports li thannek wed Melt fron I Fred Ciflerence brach E ernennen Aula Homalizalion Regin L na i n
115. er which includes a sample legend This consists of colored cells each of which corresponds to a sample in the display The toggler is used to control which samples can be seen in the display Samples with a brightly colored cell are displayed while samples with a faded cell are not displayed Samples can be switched on or off by clicking on the cell or by dragging the mouse pointer across several cells at time The Bank On and Bank Off buttons hide or display respectively all samples currently visible in the list The scroll bar can be used to display the next group of samples Note The number of displayed samples is dynamic and depends on the space available in the window Rotor Gene Q User Manual 11 2012 7 3 Analysis User Interface Clicking Named On shows only those samples that have been given a name This is a quick way to show only relevant samples Clicking All On or All Off displays all or none of the samples in the rotor respectively Pressing the Edit Samples button opens the Edit Samples window where sample names types and standard concentrations can be edited see Section 7 8 4 The toggler is shown below The additional options displayed appear after clicking the right mouse button over the toggler Page Page 1 A A A3 Ad Ah AB A AR Edit Samples Select Mon Empty Samples Toggle Sample ID Display Select Groups Bank On Bank Named All
116. er Interface 7 9 5 EE Rotor Gene Q Series Software VIRTUAL MODE no signature hrm demo analysis File Analysis Run Gain View Security Window Help o view EP of B E New Open Save tar Help Settings Progress Profle Temp Samples Analysis Reports Arrange Channels 7 Cyding A Green 2 f HRM A HRM Page Page 1 Run Settings 600nM Neat General Machine ptions Messages Channels Tube 500nM Neat General Information Run Signatures are stored within all newly saved runs These signatures like a wax seal on a document guarantee that no changes have been made outside the software If a file is tampered with the signature becomes invalid 100nM Neat 100nM Neat There is no signature 50nM Neat N There is no signature available for this run Runs saved in previous versions may d not be signed This means the file contents can not be quaranteed as free from unauthorised modification Run Signature 300nM 103 BankOn BankOff NamedOn AllOn AI Off Edit Samples OK Cancel Rotor Gene Q Series Software 47 Build 1 VIRTUAL MODE Current User CORBETT linsongy Administrators a Note If files are e mailed the encryption process can invalidate the signature To avoid this zip the file before e mailing Sample locking It is important to ensure that sample names are not accidentally or intentionally changed once a user has started a run For this reason the Rotor
117. er a defined timeout interval It is often caused by an instrument fault or by excessive activity from the PC which causes a packet to be lost Common software related causes include processor intensive tasks such as antivirus resident protection or antivirus scheduled scans wireless cards or infrared cards Disable or uninstall the relevant processor intensive software task Reset the instrument and restart the software Please contact your distributor or QIAGEN Technical Service if the problem persists This error occurs when the serial or USB communication to the instrument is lost Reconnect the serial or USB cable to the back of the computer and then click the Continue button Rotor Gene Q User Manual 11 2012 12 5 Troubleshooting Error message Object variable or with block variable not set Rotor Speed Failure Time out while setting the rotor speed Serial Port In Use The serial port is currently being used by another application Close any applications such as communications or synchronization software and then retry Shutdown timeout The instrument has exceeded the expected time to shutdown Please reset the machine and reset the software Comments and suggestions This error occurs on software startup if the default experiment template file has become corrupt This may happen if the software computer is shut down without exiting correctly for example during a power outag
118. erature Verification OTV is a method that verifies the in tube temperature in a Rotor Gene Q Validation of in tube temperature can be an important procedure in certified laboratories OTV is performed using a Rotor Disc OTV Kit see Appendix E In the following only a short introduction to the OTV principle is given Performance of the OTV procedure is explained in the Rotor Gene Q software For a more detailed description of the OTV procedure including a troubleshooting guide please refer to the Rotor Disc OTV Handbook 10 1 OTV principle OTV uses the optical properties of 3 thermochromatic liquid crystals TLC as absolute temperature references When heated TLCs change from opaque to transparent at very precise temperatures 50 C 75 and 90 TLCs do not themselves fluoresce Therefore it is necessary to cover the excitation source with a fluorescent insert so that the TLC transition points can be detected by the Rotor Gene optical system TLCs that are below their transition temperature are opaque and reflect light Some of the reflected light scatters towards the detector increasing fluorescence When the in tube temperature reaches the TLC transition point the TLC becomes transparent and light passes through the sample rather than being reflected toward the detector resulting in a decrease in fluorescence The change in fluorescence is used to determine the precise transition temperature of each TLC The tran
119. es must be in accordance with all national state and local health and safety regulations and laws 1 5 Chemicals WARNING Hazardous chemicals W12 Some chemicals used with this instrument may be hazardous or may become hazardous after completion of the protocol run Always wear safety glasses gloves and a lab coat The responsible body e g laboratory manager must take the necessary precautions to ensure that the surrounding workplace is safe and that the instrument operators are not exposed to hazardous levels of toxic substances chemical or biological as defined in the applicable Material Safety Data Sheets MSDSs or OSHA ACGIH or COSHH documents Venting for fumes and disposal of wastes must be in accordance with all national state and local health and safety regulations and laws OSHA Occupational Safety and Health Administration United States of America t ACGIH American Conference of Government Industrial Hygienists United States of America COSHH Control of Substances Hazardous to Health United Kingdom 1 6 Rotor Gene Q User Manual 11 2012 Safety Information Toxic fumes If working with volatile solvents or toxic substances you must provide an efficient laboratory ventilation system to remove vapors that may be produced 1 6 Waste disposal Used consumables and plasticware may contain hazardous chemicals or infectious agents Such wastes must be collected and disposed of prope
120. est Three windows appear the main screen the standard curve and the results Rotor Gene Q User Manual 11 2012 Analysis User Interface Reports Reports Reports opens the Report Browser window where a report of the current analysis can be generated There are 3 options standard report full report and concise report Double click on the desired option to open the report in the Preview window After the report has been generated the buttons on the top of the Preview window can be used to print save or e mail the report or export it to Word Standard Curve Std Curve This button opens the Standard Curve window By default this window is opened when an analysis is opened If you close the window it can be reopened using this command Rotor Gene Q User Manual 11 2012 7 13 Analysis User Interface M Standard Curve Cycling inml x Cycling 4 Green Page 17 F 0 99969 R 2 0 99959 M 3 292 38 949 Efficiency 1 01 Export Graph Standard Error Mean Overlay Lou Shaw Stats Zim Return to Default Position od oncentration 10 05 1006 1007 Efficiency The values on the standard curve are recalculated dynamically as the threshold level is varied by clicking and dragging the threshold line in the main window Blue dots on the curve represent the samples that have been defined as standards and red dots represent
121. etzen Sie sich mit dem Technischen Service von QIAGEN in Verbindung Falls die elektrische Sicherheit bei der Bedienung des Ger ts nicht mehr gew hrleistet werden kann muss das Ger t gegen unbefugte oder unabsichtliche Benutzung gesichert werden Kontaktieren Sie anschlie end den Technischen Service von QIAGEN Die elektrische Sicherheit des Ger ts ist nicht mehr gegeben wenn E das Ger t oder das Netzkabel besch digt erscheint E das Ger t l ngere Zeit unter ung nstigen Bedingungen gelagert wurde E das Ger t unsachgem transportiert worden ist WARNUNG Gefahr durch Stromschlag W9 Das Ger t ist mit einem Konformit tsetikett zur elektrischen Sicherheit versehen auf dem die Spannung und Frequenz der Netzstromversorgung sowie die Sicherungskennzahlen angegeben sind Das Ger t sollte nur unter diesen Bedingungen betrieben werden Rotor Gene Q User Manual 11 2012 5 Appendix C 1 3 WARNUNG A ACHTUNG A 1 4 C 6 Umgebungsbedingungen Betriebsbedingungen Explosionsf hige Atmosph ren Der Rotor Gene Q ist nicht f r den Gebrauch in explosionstahigen Atmosph ren vorgesehen Ger tesch den Direktes Sonnenlicht k nnte zum Ausbleichen von Ger teteilen f hren und Sch den an Kunststoffteilen verursachen Der Rotor Gene Q darf daher nicht in einem Bereich mit direkter Sonneneinstrahlung aufgestellt werden Biologische Sicherheit Bei Substanzen und Reagenzien die biologi
122. extfeld wie diesem beschrieben ACHTUNG Der Begriff ACHTUNG CAUTION weist Sie auf Situationen hin in denen das Ger t oder andere Ger te besch digt werden k nnten N here Einzelheiten ber diese Situationen werden in einem Textfeld wie diesem beschrieben Die in diesem Handbuch enthaltenen Hinweise stellen eine Erg nzung und keinen Ersatz der blichen Sicherheits anforderungen dar die im jeweiligen Land gelten Rotor Gene Q User Manual 11 2012 C Appendix C 1 1 Sachgem e Handhabung WARNUNG Verletzungsgefahr und Besch digung des Ger ts ACHTUNG Die unsachgem e Bedienung des Rotor Gene Q kann zu A einer Verletzung des Benutzers oder zur Besch digung des Ger ts f hren Der Rotor Gene Q darf nur durch qualifiziertes Personal das entsprechend geschult wurde bedient werden Die Instandhaltung des Rotor Gene Q darf nur durch einen Servicespezialisten des QIAGEN Au endiensts durchgef hrt werden F hren Sie alle Wartungsarbeiten gem den Anweisungen in Abschnitt 9 durch QIAGEN stellt alle Reparaturen in Rechnung die nachweislich auf eine inkorrekte Wartung zur ckzuf hren sind WARNUNG Verletzungsgefahr und Besch digung des Ger ts w23 ACHTUNG Der Rotor Gene Q ist sehr schwer Heben Sie das Ger t A vorsichtig an um eine Verletzung und oder Besch digung des Ger ts zu vermeiden WARNUNG Verletzungsgefahr und Besch digung des Ger ts ACHTUNG Bewegen S
123. file the Temperature window shows the temperature history during the run The vertical scale represents temperature and the horizontal scale represents time Use the scrollbar to scroll backwards and forwards through the Temperature window Rotor Gene Q User Manual 11 2012 7 71 Analysis User Interface 7 8 3 ini xl 00 00 00 01 00 02 00 03 00 04 Set Actual 27 8 Hold For Close Profile Progress Select Profile Progress from the View menu or click on the Progress button to bring up the Profile Progress window This window shows a graphical representation of the thermal profile associated with the run When performing a run the shaded portion of the window indicates the number of cycles that have been completed There is also an estimate of how many minutes the run will take to finish amp Profile Progress 2 Skip _Add 5 Cycles Curar running Cycling Repeat 1 Skip Skip allows any steps of the profile to be skipped 7 72 Rotor Gene Q User Manual 11 2012 Analysis User Interface 7 8 4 Add 5 Cycles Add 5 Cycles adds 5 repeats to the current cycling step Edit Samples 181 File Edit Format Security Standard Rotor Style Settings p Conc Format 123 457 Samples m gt Edit Reset Default Gradient NES FIIE E ame T ype G roups G iven Conc mem R 1000 Standard 1 000 Yes
124. g conventions i e 1 12 through to H1 H12 The additional 4 reference wells are labeled R1 R4 For more information on the Rotor Disc 100 see Section 5 3 Rotor Gene Q User Manual 11 2012 5 3 Operating Procedures Hardware 5 2 5 4 Rotor specifications Recommended Well Sample reaction Rotor type capacity no Tube type volume 36 Well Rotor 200 ul 36 PCR Tubes 20 50 ul 0 2 ml 72 Well Rotor 100 ul 72 Strip Tubes 10 50 ul and Caps 0 1 ml Rotor Disc 72 100 ul Rotor Disc 72 20 25 ul Rotor Rotor Disc 100 30 ul Rotor Disc 15 25 ul Rotor 100 Note The 36 Well Rotor and 72 Well Rotor for the Rotor Gene Q are not to be used on Rotor Gene 3000 instruments due to optical alignment incompatibilities Please continue to use the older 36 position and 72 position rotors with Rotor Gene 3000 instruments Reaction setup IMPORTANT Adequate controls should be used in each run to ensure reliable results Reactions can be prepared using the Loading Block 96 x 0 2 ml Tubes for PCR Tubes 0 2 ml the Loading Block 72 x 0 1 ml Tubes for Strip Tubes and Caps 0 1 ml set up with a single channel pipet the Loading Block 72 x 0 1 ml Multi channel for Strip Tubes and Caps 0 1 ml set up with a multichannel pipet the Rotor Disc 72 Loading Block for the Rotor Disc 72 or the Rotor Disc 100 Loading Block for the Rotor Disc 100 All blocks are made of aluminum and can be precooled The Loading Block 72 x 0 1 m
125. gli Director Business Excellence QIAGEN Instruments AG KON 00000036 B A 6 Rotor Gene Q User Manual 11 2012 Appendix A Waste Electrical and Electronic Equipment WEEE This section provides information about disposal of waste electrical and electronic equipment by users in the European Union The European Directive 2002 96 EC on WEEE requires proper disposal of electrical and electronic equipment when it reaches its end of life The crossed out wheeled bin symbol see below indicates that this product must not be disposed of with other waste it must be taken to an approved treatment facility or to a designated collection point for recycling according to local legislation The separate collection and recycling of waste electronic equipment at the time of disposal helps to conserve natural resources and ensures that the product is recycled in a manner that protects human health and the environment QIAGEN accepts its responsibility in accordance with the specific WEEE recycling requirements and where a replacement product is being supplied by QIAGEN provides free recycling of its WEEE marked electronic equipment in Europe If a replacement product is not being purchased from QIAGEN recycling can be provided upon request at additional cost To recycle electronic equipment contact your local QIAGEN sales office for the required return form Once the form is submitted you will be contacted by QIAGEN either to request fol
126. hardware 5 1 software 6 1 Optical denature cycling 6 18 Optical system 3 3 Optical temperature verification 10 1 principle 10 1 Outlier removal 7 27 Outlook 7 108 P Page 7 3 7 5 7 77 Perform last run 6 2 6 8 Port 4 8 7 10 Profile progress 7 72 Q Quantitation 7 12 D 1 Quantitation results window 7 20 Quenched FRET 6 2 Quick start wizard 6 1 R Raw channels 7 1 Reaction setup 5 4 Index 2 Rotor Gene Q User Manual 11 2012 Index Report browser window 7 9 7 13 7 43 Rotor 36 Well 5 1 72 Well 5 2 Rotor Disc 100 5 3 Rotor Disc 72 5 2 selection 6 4 6 9 specifications 5 4 types 5 1 Rotor Disc heat sealing 5 9 setup 5 9 Rotor Disc 100 5 3 Rotor Disc 72 5 2 Rotor Disc OTV Kit 10 2 Run new 7 6 open 7 7 pause 7 66 save settings 7 66 signatures 7 98 start 7 65 stop 7 66 S Safety biological 1 5 chemicals 1 6 electrical 1 4 heat hazard 1 8 mechanical hazards 1 7 proper use 1 2 samples 1 5 toxic fumes 1 7 waste disposal 1 7 Sample page suitability window 7 80 Sample types 7 75 Scaling 8 2 Scatter graph analysis 7 49 Securily 7 70 7 84 log in 7 85 Serial number 4 8 Setup window 7 9 Sicherheitshinweise 1 Slope 7 29 Software updates 4 12 version 4 9 Spanner icon 8 6 Specifications hardware A 2 optical A 3 thermal A 3 Standard curve 7 13 7 62 calculation 7 16 export 7 15 formula 7 15
127. he safety information The instructions and safety information in the user manual must be followed to ensure safe operation of the instrument and to maintain the instrument in a safe condition 2 1 About this user manual This user manual provides information about the Rotor Gene Q in the following sections 1 Safety Information Introduction General Description Installation Procedures Operating Procedures Hardware Operating Procedures Software Analysis User Interface CON OO FW h2 Additional Functions 9 Maintenance Procedures 10 Optical Temperature Verification 11 High Resolution Melt Analysis 12 Troubleshooting 13 Glossary The appendices contain the following Technical data Safety information in French and German Mathematical techniques Declaration of Conformity Rotor Gene Q accessories Liability clause Rotor Gene Q User Manual 11 2012 2 1 Introduction 2 2 General Information 2 2 1 Technical assistance At QIAGEN we pride ourselves on the quality and availability of our technical support Our Technical Services Departments are staffed by experienced scientists with extensive practical and theoretical expertise in molecular biology and the use of QIAGEN products If you have any questions or experience any difficulties regarding the Rotor Gene Q or QIAGEN products in general do not hesitate to contact us QIAGEN customers are a major source of information regarding advan
128. he instrument take care when lifting WARNING Risk of personal injury and material damage CAUTION Do not attempt to move the Rotor Gene Q during A operation CAUTION Damage to the instrument C1 Avoid spilling water or chemicals onto the Rotor Gene Q Damage caused by water or chemical spillage will void your warranty Note In case of emergency switch off the Rotor Gene Q at the power switch at the back of the instrument and unplug the power cord from the power outlet Note Do not switch off the instrument during a run except for reasons mentioned in this manual Powering off during a run has incalculable effects on sample and analysis results It is at your own risk to continue a run after it has been interrupted by powering off the instrument 1 2 Rotor Gene Q User Manual 11 2012 Safety Information WARNING Risk of personal injury and material damage 4 CAUTION Do not try to open the lid during experiment or while the Rotor Gene Q is spinning Otherwise if you overcome the lid lock and reach inside you risk contact with parts that are hot electrically live or moving at high speed and you may injure yourself and damage the instrument WARNING Risk of personal injury and material damage w5 CAUTION If you need to stop an experiment quickly turn off the power to the instrument then open the lid Let the chamber cool before reaching inside Otherwise you risk injury by touching paris th
129. he template used during Optical Temperature Verification OTV For more information see Section 10 This template is locked to ensure the profile will always operate correctly Note User defined templates can be added to the template list by copying or saving ret files to C Program Files Rotor Gene Q Software Templates After copying a file to this path the template will appear as an icon in the list New Run Wizard window 1 In the next window select the rotor type from the list Check the Locking Ring Attached checkbox and click Next to proceed Rotor Gene Q User Manual 11 2012 6 9 Operating Procedures Software New Run Wizard Welcome to the Advanced Hun Wizard Rotor Type F2 Well Rotor Rator Disc 72 Rator Disc 100 gt gt 6 2 2 New Run Wizard window 2 In the next window the user s name and notes about the run can be entered The reaction volume must also be entered If the 72 Well Rotor was selected in window 1 three Sample Layout options are available in the drop down menu 1 2 3 is the default option Most users select this option 1A 1B 1C should be selected when samples were loaded in adjacent 0 1 ml Strip Tubes using a multichannel pipet with 8 channels The A1 A2 A3 layout may be selected if appropriate 6 10 Rotor Gene Q User Manual 11 2012 Operating Procedures Software Hew Run Wizard This screen displays miscellaneous options
130. hide sample pages when they are not relevant in the current contest For example by defining a Sample Page to apply only to the Yellow channel will not be prompted to select it during an analysis of a Green channel This feature is of particular use for users of Multiplexed assays Fage Suitabilities for Selected Page C Always display this sample page 5 Only display this sample page when analysing data acquired on the following channels Green rello Orange Save amp Close Red Help Note When setting up an assay create all of the sample pages and sample page suitabilities then save them as a template This reduces the amount of setup required for each run Groups Sample groups allow statistics to be calculated for an arbitrary collection of samples Unlike replicates which must have identical names samples can have any name can be positioned anywhere in the rotor and can belong to multiple groups 1 To define a group type the full name of the group next to a sample and then press ENTER Rotor Gene Q User Manual 11 2012 7 81 Analysis User Interface BE File Edit Format Security Standard Rotor Style Settings Given Conc Format Unit Copies More Options Samples ed aaae C iD Name Type Groups Given Conc Selected Tissue Unknown A2 Tissue Unknown Tissue Unknown Ti
131. hr und Besch digung des Ger ts w5 ACHTUNG Wenn Sie ein Experiment schnell abbrechen wollen A schalten Sie das Ger t aus bevor Sie den Deckel ffnen Lassen Sie die Rotorkammer abk hlen bevor Sie ins Ger t hineingreifen Andernfalls besteht Verletzungs gefahr wenn Sie hei e Ger teteile ber hren WARNUNG Verletzungsgefahr und Besch digung des Ger ts ws ACHTUNG Bei unsachgem em vom Hersteller nicht vorgesehenem Gebrauch des Ger ts kann der vom Ger t gew hrleistete Schutz beeintr chtigt sein WARNUNG Verletzungsgefahr und Besch digung des Ger ts w7 ACHTUNG Unter dem Rotor Gene Q abgelegtes Papier st rt die K hlung des Ger ts Es wird empfohlen die Fl che unter dem Ger t frei zu halten Rotor Gene Q User Manual 11 2012 C 3 Appendix C ACHTUNG Ger tesch den C2 Verwenden Sie den Rotor immer mit einem Sicherungsring Dadurch wird vermieden dass sich die Deckel der Reaktionsgef De w hrend eines Experiments ffnen Andernfalls k nnte es zu einer Besch digung der Rotorkammer kommen wenn sich die Deckel ffnen ACHTUNG Ger tesch den C3 Kontrollieren Sie vor jedem Lauf dass der Rotor nicht besch digt oder deformiert ist Falls Sie den Rotor Gene Q w hrend eines Experiments ber hren wenn Sie elektrostatisch aufgeladen sind kann es im Extremfall zur Unterbrechung des Laufs und R cksetzen des Rotor Gene Q kommen Anschlie end wird ein Neustart der Soft
132. ie den Rotor Gene Q auf keinen Fall w hrend A des Betriebs ACHTUNG Ger tesch den C1 Vermeiden Sie es Wasser oder Chemikalien auf der Oberfl che des Rotor Gene Q zu versch tten Durch versch ttete Chemikalien oder versch ttetes Wasser verursachte Ger tesch den sind nicht durch die Garantie abgedeckt Hinweis Schalten Sie im Notfall den Rotor Gene Q aus der Netzschalter befindet sich auf der Ger ter ckseite und ziehen Sie den Netzstecker aus der Steckdose C 2 Rotor Gene Q User Manual 11 2012 Appendix C Hinweis Schalten Sie das Ger t nicht w hrend eines Laufs aus es sei denn es liegt einer der in diesem Handbuch genannten Gr nde daf r vor Das Ausschalten w hrend eines Laufs hat nicht absehbare Folgen auf Probenverarbeitung und Analyseergebnisse Wenn Sie einen Lauf nach einer Unterbrechung die durch Ausschalten des Ger ts verursacht wurde fortsetzen dann erfolgt dies auf Ihr eigenes Risiko WARNUNG Verletzungsgefahr und Besch digung des Ger ts w4 ACHTUNG Versuchen Sie nicht den Deckel des Rotor Gene zu ffnen w hrend ein Experiment l uft oder sich der Rotor noch dreht Andernfalls es wenn Sie die Deckelverriegelung gewaltsam ffnen und ins Ger te innere greifen zum Kontakt mit Teilen kommen die hei sind unter Spannung stehen oder mit hoher Drehzahl rotieren Dabei k nnten Sie sich verletzen oder das Ger t besch digen WARNUNG Verletzungsgefa
133. ification sample concentrations similar The concentration of a DNA fragment affects its melting temperature T For this reason sample DNA concentrations must be kept as similar as possible When analyzing PCR products ensure that every reaction has amplified to the plateau phase At plateau all reactions will have amplified to a similar extent irrespective of their starting amount Note however that poor reactions may not reach plateau with the same amplified quantity due for example to inconsistent assay setup e g the primer concentration was too low Ensure sample to sample uniformity All samples must be of equal volume and should contain the same concentration of dye DNA melting behavior is affected by salts in the reaction mix so it is important that the concentration of buffer Mg and other salts are as uniform as possible in all samples Similarly use only identical reaction tubes from the same manufacturer to avoid variations due to plastic thickness and autofluorescence properties 11 8 Rotor Gene Q User Manual 11 2012 High Resolution Melt Analysis Allow sufficient data collection for premelt and postmelt phases Capture HRM data points over approximately a 10 C range centered around the observed T see figure on page 11 1 This provides sufficient baseline data points for effective curve normalization and will result in more reproducible replicates and easier data interpretation 11 6 Sample pre
134. ignals with extremely high optical and thermal precision creating many application possibilities Melt curve Melt phase Postmelt phase Hamak ej 1 3 d 2 0 m TA Tg TL TBB 7 5 TRS m5 Bin BLE A typical HRM plot The melt curve plots the transition from the high fluorescence of the initial premelt phase through the decrease in fluorescence of the melt phase to the basal level of fluorescence at the postmelt phase Fluorescence decreases as DNA intercalating dye is released from dsDNA as it melts into single strands The midpoint of the melt phase at which the rate of change in fluorescence is greatest defines the melting temperature of the DNA under analysis Betore performing HRM analysis the target sequence must be amplified to a high copy number This is usually performed by PCR in the presence of a dsDNA intercalating fluorescent dye The dye does not interact with ssDNA but actively intercalates with dsDNA and fluoresces brightly when intercalated Change in fluorescence can be used to Rotor Gene Q User Manual 11 2012 11 1 High Resolution Melt Analysis measure the increase in DNA concentration during PCR and then to directly measure thermally induced DNA melting by HRM During HRM fluorescence is initially high because the sample starts as dsDNA Fluorescence decreases as the temperature is raised and DNA dissociates into single strands The observed mel
135. im Betrieb des Abzugs und bei der Entsorgung von Abfallstoffen m ssen alle Bestimmungen und Gesetze zu Gesundheitsschutz und Sicherheit am Arbeitsplatz auf bernationaler nationaler und regionaler Ebene eingehalten werden OSHA Occupational Safety and Health Administration Vereinigte Staaten von Amerika t ACGIH American Conference of Government Industrial Hygienists Vereinigte Staaten von Amerika COSHH Control of Substances Hazardous to Health Vereinigtes K nigreich Rotor Gene Q User Manual 11 2012 C 7 Appendix C 1 5 Chemikalien WARNUNG Gef hrliche Chemikalien W12 Einige Chemikalien die mit diesem Ger t verwendet werden k nnen gef hrlich sein oder nach Beendigung eines Protokolllaufs gef hrlich werden Tragen Sie immer eine Schutzbrille Laborhandschuhe und einen Laborkittel Die verantwortliche Person z B der Laborleiter muss alle erforderlichen Vorsichtsma nahmen treffen um sicherzustellen dass die unmittelbare Umgebung des Arbeitsplatzes sicher ist Auch d rfen die Grenzwerte in Bezug aut infekti se Erreger die in den entsprechenden Sicherheitsdatenbl ttern MSDS oder den Vorschriften der OSHA ACGIH oder COSHH festgelegt sind nicht berschritten werden Beim Betrieb des Abzugs und bei der Entsorgung von Abfallstoffen m ssen alle Bestimmungen und Gesetze zu Gesundheitsschutz und Sicherheit am Arbeitsplatz auf bernationaler nationaler und regionaler Ebene eingehalten werde
136. imisation amp t 60 Degrees At Beginning Of Run Channel Settings ml Add Min Gain Edit Green 1 Remove Remove All 10 Start the run by clicking Start Run and save the run file to your computer New Run Wizard Summary Green Gain fi Rotor reel Rotor Sample Layout 12253 Reaction Yolume in microliters 25 Start Hun Once you ve confirmed that your run settings are correct click Start Run to Save Template begin the run Click Save Template to save settings for future runs lt lt Back 11 16 Rotor Gene Q User Manual 11 2012 High Resolution Melt Analysis 11 8 11 Edit the sample names optional Sample names can be edited during or after a run _inix File Edit Format Security Standard Rotor Style m Settings Given Conc Format Unit copies ul More ptions Samples 0111100111210 fu nknown E 2 wild type 1 Unknown 3 wild type 1 Unknown RE 4 mutation 1 Unknown ru 5 mutation 1 Unknown xS 5 mutation 1 Unknown 7 heterozygote 1 Unknown 8 heterozygote 1 Unknown 3 heterozygote 1 Unknown Unknown 11 Unknown 12 Unknown Page Name Page 1 lt gt New Delete Synchronize pages Undo DK Cancel Real time PCR data analysis Analysis of the real time PCR data prior to HRM data analysis is advantageous Real time PCR data can highlight poorly performing assays I
137. in a shared area Run Signatures provide extra security The screenshot shows the Security tab in Run Settings for a file with a Run Signature 7 98 Rotor Gene Q User Manual 11 2012 Analysis User Interface General Machine Options Messages Channels General Information Hun Signatures stored within all newly saved runs These signatures like a was seal on a document guarantee that no changes have been made outside the software IF a File is tampered with the signature becomes invalid Hun Signature The signature is valid The signature for this run File is valid The contents has not been modified outside of the software The Run Signature is a long word that is generated each time the file is saved and linked to the contents of the file For example the signature for this file is 51758777013e2 172ef9cc9bd0c36c08 1 If the file is opened in Notepad and an edit is made e g the run date is changed to 3 days earlier the following message appears when the file is reopened Bad Run File Signature The loaded run file contains a signature which does not match the file contents This means the file has either been corrupted or tampered with since it was written by the Rater Gene 0 Seres Software Hun file signing ensures the integrity af pour run results Information about run s signature can be found in the Run Info window Rotor Gene Q User Manual 11 2012 99 Analysis Us
138. in the 36 Well Rotor Aluminum block for manual reaction setup in a standard 8 x 12 array using 96 x 0 2 ml tubes Kit for optical temperature verification of Rotor Gene systems includes a Rotor Disc preloaded with thermochromatic liquid crystals fluorescent inserts CD with calibration files requires Rotor Disc 72 Rotor and Locking Ring or Rotor Disc 72 Starter Kit Metal tree standing holder for assembling tubes and Rotor Discs into rotors Rotor Gene Q User Manual Cat no 9018901 9018902 981005 981008 9018907 9018906 9018905 981400 9018908 11 2012 Appendix E Product Contents Cat no QIAGEN HRM and quantitative real time PCR kits recommended for use with the Rotor Gene Q Rotor Gene SYBR For 400 x 25 ul reactions 3 x 1 7 ml 2x 204074 Green PCR Kit 400 Rotor Gene SYBR Green PCR Master Mix 2 x 2 ml RNase Free Water Rotor Gene SYBR For 400 x 25 ul reactions 3 x 1 7 ml 2x 204174 Green RT PCR Kit Rotor Gene SYBR Green RT PCR Master 400 Mix 100 ul Rotor Gene RT Mix 2 x 2 ml RNase Free Water Rotor Gene Probe For 400 x 25 ul reactions 3 x 1 7 ml 2x 204374 PCR Kit 400 Rotor Gene Probe PCR Master Mix 2 x 2 ml RNase Free Water Rotor Gene Probe For 400 x 25 ul reactions 3 x 1 7 ml 2x 204574 RT PCR Kit 400 Rotor Gene Probe RT PCR Master Mix 100 ul Rotor Gene RT Mix 2x2 ml RNase Free Water Rotor Gene Multiplex For 400 x 25 ul reactions 3 x 1 7 ml 2x 204774 PCR Kit 40
139. including but not limited to 5 nuclease assays or to any patent claiming a reagent or kit For further information on purchasing additional rights contact the Director of Licensing at Applied Biosystems 850 Lincoln Centre Drive Foster City California 94404 USA For applicable countries The purchase of this product includes a limited non transferable license to one or more of US Patents Nos 6 787 338 7 238 321 7 081 226 6 174 670 6 245 514 6 569 627 6 303 305 6 503 720 5 871 908 6 691 041 7 387 887 and U S Patent Applications Nos 2003 0224434 and 2006 0019253 and all continuations and divisionals and corresponding claims in patents and patent applications outside the United States owned by the University of Utah Research Foundation Idaho Technology Inc and or Roche Diagnostics GmbH for internal research use or for non in vitro diagnostics applications No right is conveyed expressly by implication or estoppel for any reagent or kit or under any other patent or patent claims owned by the University of Utah Research Foundation Idaho Technology Inc and or Roche Diagnostics GmbH or by any other Party For information on purchasing licences for in vitro diagnostics applications or reagents contact Roche Molecular Systems 4300 Hacienda Drive Pleasanton CA 94588 USA For up to date licensing information and product specific disclaimers see the respective QIAGEN kit handbook or user manual QIAGEN kit handbooks and user manua
140. ing A Green Page 1 ioj x 9 Reports Results Adjust Scale Auto Scale Default Scale Comp Quant Results Cycling A Green Page 1 _ 1501 x Take Off Amplification Comparative Conc Rep Takeoff Rep Takeoff 95 Rep Amp Rep Amp 95 Rep Conc Rep C 1 mutation 1 11 2 1 66 1 00E 00 11 2 1 66 1 00 00 Calibra 2 mutation 2 11 4 1 65 9 04 01 11 4 1 65 9 04E 01 3 mutation 3 11 4 1 64 3 04E 01 11 4 1 64 3 04E 01 4 wildtype 1 11 9 1 66 7 01E 01 11 9 1 66 7 01E 01 5 wild type 2 11 8 1 66 7 38E 01 11 8 1 66 7 38E 01 6 wild type 3 11 9 1 66 7 01E 01 11 9 1 66 7 01E 01 7 heterozygote 1 11 4 1 67 9 04 01 11 4 1 67 9 04E 01 8 heterozygote 2 11 5 1 66 8 59 01 11 5 1 66 8 59 01 9 heterozygote 3 11 3 1 67 9 51E 01 11 3 1 67 9 51E 01 4 Comparative quantitation results The reaction efficiency is shown in the Amplification column as a score out of 2 2 100 efficiency Note If you suspect the presence of primer dimers or nonspecific products assess reactions by drawing a derivative plot using the Melt option from the Analysis window Ensure that there is a single peak indicative of a single product If possible run a gel to check that there is a single amplification product If there is more than one product the reaction should be repeated or reoptimized 11 18 Rotor Gene Q User Manual 11 2012 High Resolution Melt Analysis 11 9 HRM data anal
141. ing representatives of QIAGEN which are inconsistent or in conflict with the conditions in this warranty shall not be binding upon the Company unless produced in writing and approved by an officer of QIAGEN Rotor Gene Q User Manual 11 2012 F 1 Appendix F This page intentionally left blank F 2 Rotor Gene Q User Manual 11 2012 Index Index A Acquisition 6 15 Adiust scale 7 2 Advanced wizard 6 7 Allelic discrimination 7 47 Audit trails 7 95 Auto find threshold 7 20 Autoscale 7 2 AutoStat 7 24 C Calibrator replicate 7 46 Cautions 1 1 Channels 3 4 7 68 Comparative quantitation 7 44 Concentration analysis 7 61 Confidence intervals D 2 concentrations 0 1 Correlation coefficient 7 15 Crop cycles 7 3 C calculation 7 18 Ct Comment 7 21 Cycling 6 14 long range 6 15 touchdown 6 15 D Default scale 7 2 Delta delta relative quantitation 7 36 Detection parameters 3 4 Dynamic tube normalization 7 26 7 50 Edit profile window 6 5 6 12 Edit samples window 6 7 6 31 7 73 rotor style 7 79 Efficiency 7 14 7 29 Empty run 6 8 EndPoint analysis 7 52 controls 7 56 Error message 12 3 Excitation parameters 3 4 Exponential amplification 7 29 Export data 8 5 graphs 8 2 native format 8 4 to LinReg 7 8 F Fluorophores detected 3 4 Fumes toxic 1 7 G Gain optimisation 6 11 6 24 manual 6 29 Gain settings 7 103 Genotypes allelic discrimination
142. instrument to acclimatize to room temperature for at least an hour before turning the instrument on If the error persists please contact your distributor or QIAGEN Technical Service This error occurs when the software has detected a fatal hardware malfunction and has activated a safe protection procedure to turn off the machine Turn off the instrument immediately and contact your distributor or QIAGEN Technical Service 12 4 Rotor Gene Q User Manual 11 2012 Troubleshooting Error message Machine Error This run was stopped as machine errors occurred that could not be recovered from Please contact your distributor if this occurs again attaching a support archive file Machine Unplugged The instrument is not responding and failed with the message lt ERROR MESSAGE gt This is an unrecoverable failure please reset the instrument and restart the software Machine Unplugged The instrument is not connected to your computer on lt PORT NAME gt Reconnect the serial cable to the back of the computer and then click Continue Comments and suggestions This error occurs when the software has detected errors on the machine that could not be recovered from The software has stopped the run Try another run If the problem persists contact your distributor or QIAGEN Technical Service and attach a support archive file This error occurs if the instrument does not communicate with the software aft
143. isition channels to be specitied The initial protile shown is based on the template selected when setting up the run see page 6 1 The protile is displayed graphically The list of the segments of the profile appears below the graphical display This list can include Hold page 6 13 Cycling page 6 14 Melt page 6 17 or HRM if the instrument has a HRM channel page 6 18 Each stage of the profile can be edited by clicking on the appropriate area of the graphical display or on the name in the list and then changing the settings which appear Insert after This allows addition of a new cycle after the selected cycle Insert before This allows addition of a new cycle before the selected cycle Remove This removes the selected cycle from the profile 6 12 Rotor Gene Q User Manual 11 2012 Operating Procedures Software Hold A Hold instructs the Rotor Gene Q to remain at the designated temperature for a set time To change the temperature click on the Hold Temperature button and type or use the slide bar to select the desired temperature To change the duration of the Hold click on the Hold Time mins and secs buttons x E 3 Mew Open Save As Help The run will take approximately 89 minute s to complete The graph below represents the run to be performed Click on a cycle below to modify it Insert after Insert before Remove Hold Temperature
144. it is not sufficient to double click on one channel to analyze because this analysis is performed using multiple channels simultaneously To perform this analysis either hold down CTRL and click to highlight each channel you wish to analyze or drag the mouse pointer over these channels Once the desired channels have been highlighted click Show The list will update to show all the channels on one line with a checkmark beside them This indicates that they will all be used in one analysis To remove one or more of these channels right click on the analysis and select Rotor Gene Q User Manual 11 2012 7 47 Analysis User Interface Remove Analysis Those channels can then be included in another allelic discrimination analysis A channel can only be used in one analysis at a time Reports Results Normalization options Discrimination Threshold This opens the Allelic Discrimination Analysis report for preview This displays the Allelic Discrimination Results window This window is opened by default when the analysis is first displayed A variety of options are available to optimize raw data normalization E Dynamic Tube dynamic tube normalization E Slope Correct noise slope correction M Ignore First x cycles correction for noise in initial cycles For more details see page 7 25 Enter values in these text boxes to position the discrimination threshold All curves passing this threshold ar
145. ize Note the end point fluorescence Fl reading If the Fl reading is not at the required level click on Edit Gains and edit as required This process may not be instantaneous as the Rotor Gene Q takes 4 seconds to acquire each point in each channel and during this time the user interface is deactivated Repeat the process until the Fl is at the desired level Click on Stop If the run is still acquiring data when the Stop button is clicked the Rotor Gene Q finishes acquiring first and then stops This process can take up to 5 seconds for each acquiring channel Rotor Gene Q User Manual 11 2012 Operating Procedures Software 6 2 5 6 2 6 New Run Wizard window 4 This window summarizes the run Check the parameters and If they are correct click Start Run You will be prompted for a file name You can also save the run settings as a template for future runs using the Save Template button Hew Run Wizard Summary Setting Green Gain 5 Rotor 36 w ell Rotor Sample Layout 1 2 3 Reaction Yolume in microliters 25 Start Aun Once you ve confirmed that your run settings are correct click Start Run to Save Template begin the run Click Save Template to save settings for future runs Skip Wizard lt lt Back New Run Wizard window 5 Enter sample types and descriptions in this window while the run is in progress The functionality of this window is identical to the
146. l Tubes pictured holds 18 Strip Tubes as well as up to eight 0 5 ml tubes which can be used to prepare master mix and up to sixteen 0 2 ml tubes Rotor Gene Q User Manual 11 2012 Operating Procedures Hardware which can be used to set up standard curves The procedure below describes reaction setup using the 72 Well Rotor The same procedure can be used for reaction setup using the 36 Well Rotor and appropriate accessories 1 Place the Strip Tubes into the Loading Block and aliquot the reaction components 2 Place the Caps securely on the Strip Tubes and visually inspect to confirm a tight seal Rotor Gene Q User Manual 11 2012 5 5 Operating Procedures Hardware 3 Insert the Strip Tubes into the 72 Well Rotor ensuring that each tube sits correctly in place Samples will not be optimally aligned over the detection system if not placed correctly in the rotor This could result in a reduction in acquired fluorescence signal and detection sensitivity A Rotor Holder that enables easy tube loading is provided with the instrument 5 6 Rotor Gene Q User Manual 11 2012 Operating Procedures Hardware IMPORTANT To achieve maximum temperature uniformity each position in the rotor must contain a tube Filling all positions in the rotor ensures even airflow to every tube Keep a set of empty capped tubes available that can be used to fill any unused positions 4 Insert the 72 Well Rotor Locking Ring on
147. les the windows to be tiled vertically or horizontally or arranged in a cascade Further options are accessible by clicking the arrow on the right of the Arrange button Help function When using the Help button or Help menu the following drop down menu will open x Hel Contents What s Her Change History Website Send Support Email About This Software Choose the desired options for details Send Support E Mail The Send Support Email option in the Help menu allows you to send a support email to QIAGEN including all relevant information from a run The Save As option will save all the information to a file that you can copy onto a disk or across a network if you do not have access to e mail on the computer running the Rotor Gene Q If you use the support e mail function on the laptop computer provided optionally with the Rotor Gene Q country dependent for the first time you have to configure your e mail settings Note You can make the entries of the IT manager of your company 7 104 Rotor Gene Q User Manual 11 2012 Analysis User Interface Configure the e mail settings Click the option Send Support Email The following window will open Internet Connection Wizard X Tour Name When vou send e mail your name will appear in the From Field of the autgaing message Type your name as you would like it to appear Display name For example Jahn Smith 1
148. lez ces chantillons avec la plus grande pr caution et conform ment aux r gles de s curit n cessaires Portez toujours des lunettes de protection deux paires de gants et une blouse de laboratoire La personne responsable par exemple le directeur du laboratoire doit prendre les pr cautions n cessaires afin de garantir que l environnement de travail est s r que les op rateurs de l appareil sont convenablement form s et ne sont pas expos s des niveaux dangereux d agents infectieux comme cela est d fini dans les fiches techniques sant s curit MSDS ou dans les documents de l OSHA de l ACGIH ou du COSHH applicables L vacuation des vapeurs et la mise au rebut des d chets doivent s effectuer conform ment toutes les r gles et lois nationales r gionales et locales relatives la sant et la s curit OSHA Occupational Safety and Health Administration tats Unis d Am rique administration pour la sant et la s curit du travail ACGIH American Conference of Government Industrial Hygienists tats Unis d Am rique Conf rence am ricaine des hygi nistes industriels gouvernementaux COSHH Control of Substances Hazardous to Health Royaume Uni contr le des substances dangereuses pour la sant Rotor Gene Q User Manual 11 2012 B 7 Appendix B 1 5 Produits chimiques AVERTISSEMENT Produits chimiques dangereux W12 Certains produits chimiques utilises ave
149. lines can be emphasized by modifying their style To access this feature click on the right arrow button next to the Edit button E dit Reset Default Gradient 7 78 Rotor Gene Q User Manual 11 2012 Analysis User Interface The toolbar will show the default style Solid This can be changed to Dashed Dotted Hairline Thin or Thick When finished click the left arrow button to return to the Edit Reset Default and Gradient view sn Multiple row If the same information needs to be entry entered in several rows at once select all the rows then begin to type The information will be entered into each row This also works for selecting sample types choosing colors or entering concentrations c alid Sample type To quickly select a sample type enter the hotkey first letter of its name For example to set 5 samples to be no template controls select them in the sample type column then press N for NTC All samples will be converted to NTC Save it reuse it A complete sample description can be saved as a sample file smp and loaded into future runs with the same sample configuration Rotor Style This tab in the Edit Samples window provides an alternative way of entering sample names Select replicates by clicking and dragging the mouse pointer over the rotor image The list on the right of the window will update The sample name can be typed in and this will set the same n
150. ll be detected automatically and displayed in the Port drop down list 4 8 Rotor Gene Q User Manual 11 2012 Installation Procedures 4 8 Run in Virtual Checking this box allows installation of the Mode Rotor Gene Q software on a computer that for is not connected to a Rotor Gene Q The demonstration software is fully functional and can simulate runs Note If this box is checked and a Rotor Gene Q is connected to the computer the following message appears before the run starts You are about to run in Virtual mode To perform a real run the setup must be changed in the Setup window see Section 7 5 4 Begin When all the information has been entered click Begin Wait until initialization is finished which may take a few seconds If virtual mode was chosen the following message appears Rotor Gene Q Series Software x i This software will perform basic simulation of a machine For training and demonstration purposes You can disable this setting via the Setup screen accessible From the File menu If the Run in Virtual Mode box is unchecked the software initializes and opens automatically Exit Program Clicking on this button exits the program Software version To find out the Rotor Gene Q software version number click on Help then About This Software The About This Software window displays general information about the software including the version of
151. low up information for scheduling collection of the electronic waste or to provide you with an individual quote Rotor Gene Q User Manual 11 2012 A 7 Appendix A This page intentionally left blank A 8 Rotor Gene Q User Manual 11 2012 Appendix B Appendix B Safety Information French FR 1 Informations de s curit Avant d utiliser le Rotor Gene Q il est imp ratif de lire attentivement ce manuel et de porter une attention particuli re aux informations de s curit Afin de garantir un fonctionnement de l appareil en toute s curit et de maintenir l appareil en bon tat de marche il est imp ratif de suivre les instructions et les informations de s curit fournies dans le pr sent manuel d utilisation Les types d informations de s curit suivants sont fournis tout au long du manuel AVERTISSEMENT Le terme AVERTISSEMENT signale des situations risquant d entrainer des accidents corporels dont vous ou d autres personnes pourriez tre victime Les d tails concernant ces circonstances sont donn s dans un encadr identique celui ci ATTENTION Le terme ATTENTION signale des situations risquant d entra ner des d t riorations de l appareil ou de tout autre mat riel Les d tails concernant ces circonstances sont donn s dans un encadr identique celui ci Les conseils donn s dans ce manuel ont pour but de venir compl ter les exigences de s curit habituelles en vigueur dans le pays de
152. ls are available at www qiagen com or can be requested from QIAGEN Technical services or your local distributors 2005 2012 QIAGEN all rights reserved Contents Contents 1 Safety Information Proper use Electrical safety Environment Biological safety Chemicals Waste disposal Mechanical hazards Heat hazard 0 0 KR M Maintenance O Symbols on the Rotor Gene Q 2 Introduction 2 1 About this user manual 2 2 General Information 2 2 1 Technical assistance 2 2 2 Policy statement 2 2 3 Version management 2 3 Intended use of the Rotor Gene Q 3 General Description 3 1 Thermal performance 3 2 Optical system 4 Installation Procedures 4 Site requirements 4 2 Power connection Rotor Gene Q User Manual 11 2012 1 1 1 2 1 4 1 5 1 5 1 6 1 7 1 7 1 8 1 9 1 10 2 1 2 1 2 2 2 2 2 2 2 2 2 3 3 1 3 1 3 3 4 1 4 4 2 Contents 1 Contents 4 3 PC requirements 4 2 4 4 Unpacking the Rotor Gene Q 4 3 4 5 Accessories 4 4 4 6 Hardware installation 4 4 4 7 Software installation 4 6 4 8 Software version 4 9 4 9 Additional software on connected computers 4 10 4 9 1 Virus scanners 4 11 4 9 2 System tools 4 11 4 9 3 Operating system updates 4 12 4 10 Updating software 4 12 5 Operating Procedures Hardware 5 1 5 1 Rotor types 5 1 5 2 Reaction setup 5 4 5 3 Rotor Disc setup 5 9 6 Operating Procedures Software 6 1 6 1 Quick Start wizard 6 1
153. lysis User Interface channels Cycling A Green and Cycling A Yellow cross the threshold EndPoint analysis templates EndPoint analysis templates allow the user to export genotype and threshold settings into a single ent file This file can be imported and reapplied in other experiments See Section 8 1 for more details Imported Settings Import Export 7 6 10 Concentration analysis Concentration analysis allows the Rotor Gene Q to be used to measure DNA concentrations or to obtain fluorometer readings The screenshot below shows this analysis EU Rotor Gene Q Series Software VIRTUAL MODE DNA concentration File Analysis Run Gain View Security Window Help d 3 B X New Open Save 2805 Help Settings Progress Profile Temp Samples Analysis Reports Arrange Channels 7 Cyding A Green 5 1 El Concentration Measurement Cycling A Green Page 1 BAR _ 2 Lambda Standard 1 N c Lambda Standard 1 Reports Std Curve Results 25 00 are Cycling A Green Circles J O 4 wh b Bank On Bank Off On AllOn Al Off Edit Samples Analysis Method C Linear Regression 1 2 3 9 10111314 15 17 18 19 21 22 23 25 26 27 29 30 31 33 34 35 37 38 39 41 42 43 45 4647 Sample Spine Curve Fit Lambda Standard Standard Lambda Standard Standard Lambda Standard Standard Lambda Standard Standard 10 Lambda Standard Standard R
154. multaneously To create a new analysis click on the EndPoint tab select the channels by dragging over them with the mouse pointer and then click Show Rotor Gene Q User Manual 11 2012 7 55 Analysis User Interface Quantitation Scatter EndPoint Other Cycling Green Cycling 4 yellow Show Hide Auto shrink window Define controls When an EndPoint analysis is opened for the first time the following message will be displayed if positive and negative controls have not been defined Rotor Gene Q Series Software 1 x 1 use end point analysis you must have positive and negative controls in each channel To define these controls dick OK Click The Edit Samples window appears allowing positive and negative controls to be defined To define a sample as a positive or negative control click on the sample type cell then select the relevant control type from the drop down menu Note The controls must be toggled on using the toggler at the right hand side of the main window to perform the analysis 7 56 Rotor Gene Q User Manual 11 2012 Analysis User Interface ts Select Positive and Negative Controls Please select at least one positive and negative control then click OK These controls will be used as references for the fluorescence required for a reaction You should select positive and negative controls for each channel
155. n OSHA Occupational Safety and Health Administration Vereinigte Staaten von Amerika t ACGIH American Conference of Government Industrial Hygienists Vereinigte Staaten von Amerika COSHH Control of Substances Hazardous to Health Vereinigtes K nigreich Giftige D mpfe Alle Arbeiten mit fl chtigen L sungsmitteln oder toxischen Substanzen m ssen unter einem funktionierenden Laborabzugssystem durchgef hrt werden damit die m glicherweise entstehenden D mpfe abziehen k nnen 1 6 Entsorgen von Abf llen Benutzte Verbrauchs und Kunststoffartikel k nnten gef hrliche Chemikalien oder infekti se Erreger enthalten Derartige Abf lle m ssen gesammelt und gem den geltenden kommunalen Sicherheitsbestimmungen entsorgt werden C 8 Rotor Gene Q User Manual 11 2012 Appendix C 1 7 Gefahren durch mechanische Teile Der Deckel des Rotor Gene Q muss w hrend des Betriebs geschlossen sein WARNUNG Sich bewegende Ger teteile W13 Um einen Kontakt mit sich bewegenden Teilen beim Betrieb des Rotor Gene Q zu vermeiden darf das Ger t nur mit geschlossenem Deckel betrieben werden WARNUNG Verletzungsgefahr und Besch digung des Ger ts wi4 ACHTUNG Offnen und schlie en Sie den Deckel des Rotor Gene Q vorsichtig um das Einklemmen von Fingern oder Kleidungsteilen zu vermeiden ACHTUNG Ger tesch den C5 Stellen Sie sicher dass der Rotor und der Sicherungsring ordnungsgem eingese
156. n explosive atmosphere Damage to the instrument C4 Direct sunlight may bleach parts of the instrument and cause damage to plastic parts The Rotor Gene Q must be located out of direct sunlight Biological safety Specimens and reagents containing materials from biological sources should be treated as potentially infectious Use safe laboratory procedures as outlined in publications such as Biosafety in Microbiological and Biomedical Laboratories HHS www cdc gov od ohs biosfty biosfty htm Samples Samples may contain infectious agents You should be aware of the health hazard presented by such agents and should use store and dispose of such samples according to the required safety regulations Rotor Gene Q User Manual 11 2012 1 5 Safety Information WARNING Samples containing infectious agents w11 Some samples used with this instrument may contain A infectious agents Handle such samples with the greatest of care and in accordance with the required safety regulations Always wear safety glasses 2 pairs of gloves and a lab coat The responsible body e g laboratory manager must take the necessary precautions to ensure that the surrounding workplace is safe and that the instrument operators are suitably trained and not exposed to hazardous levels of infectious agents as defined in the applicable Material Safety Data Sheets MSDSs or OSHA ACGIH or COSHH documents Venting for fumes and disposal of wast
157. n may not be representative of the rest of the run For this reason better results may be achieved if the first few cycles are ignored However if the first cycles look similar to subsequent cycles better results will be achieved by deselecting Ignore First because the normalization algorithm will have more data to work with Outlier Removal To distinguish between minor changes in fluorescence and genuine reactions in no template controls NTCs 2 measures are provided NTC Threshold and Reaction Efficiency Threshold The NTC Threshold is recommended for most applications The approach used should be validated x NTE Threshold Percentage of largest Fl change LE 15 30 Any sample with a total change in fluorescence 2 X less than this percent relative to the largest change in any tube will not be displayed Reaction Efficiency Threshold Enabled Only samples which have an individual reaction ethency at least equal to this Threshold ou value willbe displayed after Fe normalisation Cancel Hep Rotor Gene Q User Manual 11 2012 7 27 Analysis User Interface NTC Threshold Reaction Efficiency Threshold This allows samples or NTCs that have a slight drift upwards to be excluded from analysis All samples with a change below the Threshold will not be reported and a flag NEG NTC will be shown in the CT Comment column The per
158. n on the derivation of the Rel Min and Rel Max calculations refer to Litvak and Schmittgen 2001 Relative Quant Analysis Eapgenmaar Inkemasen Gene Expression Graph Logar emkch I oh 7 6 5 Melt curve analysis Melt curve analysis analyzes the derivative of the raw data after smoothing This analysis is commonly used for genotyping and allelic discrimination Peaks in the curve are grouped into bins and all peaks below the threshold are discarded Bins can then be mapped to genotypes using the Genotypes command Livak K J and Schmittgen T D 2001 Analysis of relative gene expression data using real time quantitative PCR and the 2 delta delta C T method Methods 25 402 7 40 Rotor Gene Q User Manual 11 2012 Analysis User Interface After a run has finished for some chemistries a melt step can be added to visualize the dissociation kinetics of the amplified products The temperature is increased at a linear rate and the fluorescence of each sample is recorded A typical melt curve analysis is shown below ober Gene E Series Software Fle Analysis Run Gan View Window Help CU HNH New Oper Seve Gert Parce 12 cres Me AOL MAO M5 Ninh M5 Din 0 107 Mind 302 Din C IMn 302 Un C 805 Una 80 318 Aj Rotor Gene Q Senes Software 1 7 54 Qa rotor Gene Q Series S UT OAZ eem Feak Calling M Flip sign of dFdT Th
159. nce the standard curve has been imported the standard curve type changes to Fixed Click Reset to change the standard curve type back to Floating A screenshot of the Import Standard Curve window is shown below Import Standard Curve Import Standard Curve Current Run From Other Run Channel Cycling A Yellow 10 0 307 CT 12 525 CT 3 32z lpg conc 41 608 Cycling Orange conces 10 0 309 11 185 CT 3 235loa conc 36 187 Cycling amp Red conc 10 0 316 CT 11 817 CT 3 155 log canc 37 404 C From Extemal Source Post 6 0 Standard Curve Format CT 8B Ing Conc y Efficiency From Extemal Source Pre 6 0 Standard Curve Format Cones 10 SCS ance Efficiency Help Rotor Gene Q User Manual 11 2012 7 17 Analysis User Interface Using this window a standard curve can be imported from another channel analyzed in the current run or from another run Current Run From Other Run Channels From External Source C Calculation Invert raw data When this option is selected quantitation analyses on other channels from this run are listed with the corresponding standard curves Selecting this option brings up a dialog from which a run file can be selected to open If any quantitation analysis has been performed for the run standard curves are listed for each channel analyzed Note Th
160. ncrements of 1 C waiting for 5 seconds before each acquisition The Rotor Gene Q can be configured to perform melts in 0 02 C increments The minimum hold time between temperature steps varies depending on the number of degrees between each step High Resolution Melt High resolution melt HRM analysis characterizes double stranded DNA samples based on their dissociation melting behavior It is similar to classical melting curve analysis but provides far more information for a wider range of applications Samples can be discriminated according to sequence length GC content or strand complementarity down to single base pair changes HRM analysis can only be performed on instruments that have HRM hardware and software installed Data is acquired using specialized HRM sources and detectors HRM analysis also includes the option to perform Gain Optimisation just before the Melt begins After performing HRM the data can be analyzed with HRM analysis software Section 11 Optical Denature Cycling Optical Denature Cycling is an exciting technique available on the Rotor Gene Q which performs real time melt analysis to determine the melt peak of a reference sample This indicates PCR product denaturation with higher precision than setting a particular denature temperature for a hold time To perform this technique simply place a reference tube of PCR product in tube position 1 of the rotor The 6 18 Rotor Gene Q User Manual 11
161. ne Q User Manual 11 2012 Optical Temperature Verification 5 The run performs a series of melts that determine the thermal characteristics of the Rotor Gene Q i OTY Temperature Verification Results E X Summar Yerfhcaton Result Adjustment Recommended Details Lower Pont No Adjustment Required Middle Pont No Adjustment Required Upper Point Adjustment Required Apply Adjustment 6 When the run is finished the software indicates whether the Rotor Gene Q is within specification 7 If adjustment is required the user must click Apply Adiustment This prompts the user to perform a verification run After the verification run is complete no adjustment should be required If further adjustment is required contact your distributor 8 When the Rotor Gene Q is within specification a report of the run can be reviewed and printed Rotor Gene Q User Manual 11 2012 10 5 Optical Temperature Verification This page intentionally left blank 10 6 Rotor Gene Q User Manual 11 2012 High Resolution Melt Analysis 11 High Resolution Melt Analysis High resolution melt HRM analysis is an innovative technique that is based on analysis of DNA melting HRM characterizes DNA samples according to their dissociation behavior as they transition from double stranded DNA dsDNA to single stranded DNA ssDNA with increasing temperature see Figure below A HRM instrument collects fluorescent s
162. ne Q User Manual 11 2012 7 59 Analysis User Interface still be defined as a positive control Since the highest positive control in each channel is used if there is least one positive control in the yellow channel that amplifies the definition of the sample as a control for the green channel is ignored Threshold The threshold is used to determine the percentage expression required for a reaction in each channel Once the positive and negative controls have been defined all channels will be normalized to the same 0 100 scale For this reason only one threshold is needed even when analyzing multiple channels Click and drag the threshold line to an area between O and 100 The threshold should not be too close to samples on either side of the line because this indicates that the run was not conclusive If the difference between a sample being defined as amplified or not amplified is just a few percent this means that if the reaction were repeated the sample could appear on the other side of the threshold Genotypes This option opens the Genotyping window which is used to define which genotype is detected in each channel 5 Genotyping Reacting Channels Wild Type Cycling A Green Heterozygous Cycling 4 Green Cycling Mutant Cycling A yellow This window allows genotypes to be assigned to channels In the example above a sample is heterozygous if readings in 7 60 Rotor Gene Q User Manual 11 2012 Ana
163. nel and sample page The sample number The defined individual sample graph color The defined sample type The determined C value An automatic annotation of the C determination if C values are excluded The following flags are possible NEG Multi Ct The threshold crosses the fluorescence curve at least two times double intersect An unambiguous value cannot be determined NEG NTC The overall fluorescence increase does not fulfill the conditions defined in NTC threshold function of the Outlier Removal menu see below For example a fluorescence curve intersects with the given threshold but the minor overall slope increase suggests a non template control and a value is not given NEG R Eff The overall fluorescence increase does not fulfill the conditions defined in Reaction efficiency threshold function of the Outlier Removal menu see below Samples that do not have a certain reaction efficiency are excluded and the value is not given This flag is only shown if the corresponding function is enabled Rotor Gene Q User Manual 11 2012 7 21 Analysis User Interface Var Rep Cr Rep Std Dev Rep Cr 95 C l Rep Calc Conc The percentage variation between the calculated and the known concentration Var Abs Calculated Given 1 The average of all samples with the same name as this sample The standard deviation of the C value
164. ning as an administrator all the software features are enabled Therefore running as an administrator ensures that users who do not need security features can access all the software features Creating a new user account Create user accounts for each user of the software For each user repeat the steps below until all accounts have been created 1 To create a new user select Start Settings Control Panel 2 Double click on Users and Passwords 7 86 Rotor Gene Q User Manual 11 2012 Analysis User Interface Users and Passwords Users Advanced computer and ko change passwords and other settings G Use the list below to grant or deny users access to your Users For this computer STEW Admini trators W Guest STEW Guests USA NEWLAPTOP STEW Guests NEW LAPTOP STEW Guests matthew STEW Administrators Add Remove Properties Password For Administrator To change the password for Administrator click Set feb Password Set Password Rotor Gene Q User Manual 11 2012 7 87 Analysis User Interface 3 Click the Advanced tab then click the Advanced button Users and Passwords 1 Users Advanced Certificate Management Use certificates to positively identify Yourself certification authorities and publishers Mew Certificate Certificates Advanced User Management Local Users an
165. ns Size a Metatile width 833 as PostScript EN Height 1540 EX M Keep aspect ratio 4 Check the Keep aspect ratio checkbox to keep the image in the correct proportion when adjusting its size 5 Click Save and select a filename and location for the file in the dialog box which appears If a higher resolution image is required we recommend either increasing the size of the image until it meets your requirements or saving the graph as a Metafile emf Rotor Gene Q User Manual 11 2012 8 3 Additional Functions wmf This is a vector based format that can be opened in software such as Adobe Illustrator allowing the user to create an image of any resolution Native format export Graphs in the Rotor Gene Q software use the third party TeeChart component developed by Steema software To save a graph in native format select the Native tab in the Export Dialog window see previous screenshot and then click Save Native format is the standard TeeChart file format This allows the user to use TeeChart Office from Steema software TeeChart Office is available as freeware and is installed as part of the Rotor Gene Q software package To access the software click on the TeeChart icon on the desktop TeeChart Office enables manipulation of exported graphs including changing colors of curves adding annotations changing fonts and adjusting data points amp TeeChart Office tez
166. nual 11 2012 Installation Procedures 4 9 1 4 9 2 Virus scanners We are aware of the threat that viruses cause to any computer that exchanges data with other computers Rotor Gene sofiware is primarily installed in environments where local policies exist to minimize this threat These policies usually require use of a particular anti virus tool Due to the sheer number of anti virus tools available it is not possible for QIAGEN to predict the possible impact on the Rotor Gene Q software if such a tool is active during a PCR run In order to get consistent results system administrators should therefore ensure that during performance of a PCR run M File access is not intercepted by a virus scanner M Updates to the virus database are not performed M File scans are not performed We strongly recommend disabling virus scanner activity during real time PCR data acquisition The critical virus scanner tasks described above can only be safely carried out without running the risk of impacting the performance of the instrument when the Rotor Gene Q software is not running Otherwise there is a risk of adverse impact on the performance of the instrument System tools Many system tools may use significant system resources even without any user interaction Typical examples of such tools are M File indexing which is performed as a background task by many contemporary office applications M Disk defragmentation which often also
167. of all samples with the same name as this sample A C range that statistically accounts for 95 of the variation in the C value This is a conservative statistical measure which can be used as a quality measure This range can be narrowed by running more replicates or by having less variation in the replicates The calculated concentration for all the samples with the same name Note This is not the simple average of the calculated concentrations It is the geometric mean which is a mathematically more appropriate average due to the exponential nature of real time amplification 7 22 Rotor Gene Q User Manual 11 2012 Analysis User Interface Rep Calc Conc A range of concentrations that accounts for 95 C l 95 of the variation in the individual sample as well as the linear regression model on which it is based An interpretation of this measure is that it is the range of concentrations that could be expected 95 of the time if this run were performed repeatedly with the same amount of variation This is a conservative estimate and the range can be quite large due to the variation inherent in any real time analysis This range can be large if standards are run with concentrations different from the unknown samples if a small number of replicates are used or if there is significant variation IMPORTANT The variations reported by this measure are inherent to the exponential process of real time amplifi
168. on 3 4 Rotor Gene Q User Manual 11 2012 Installation Procedures WARNING A WARNING A Installation Procedures Site requirements Rotor Gene Q instruments must be located out of direct sunlight away trom heat sources and away from sources of vibration and electrical interference Refer to Appendix A for the operating conditions temperature and humidity The installation site should be free of excessive drafts excessive moisture excessive dust and not subject to large temperature fluctuations Refer to Appendix A for the weight and dimensions of Rotor Gene Q instruments Ensure that the workbench is dry clean and has additional space for accessories For further information about required specifications of the workbench contact QIAGEN Technical Services Note It is extremely important that the Rotor Gene Q instrument is placed on a stable surface which is level and vibration free Refer to operating conditions see Appendix A The Rotor Gene Q instrument must be placed within approximately 1 5 m 59 in of a properly grounded earthed AC power outlet Explosive atmosphere w10 The Rotor Gene Q instrument is not designed for use in an explosive atmosphere Risk of overheating W16 To ensure proper ventilation maintain a minimum clearance of 10 cm 3 94 in at the rear of the Rotor Gene Q instrument Slits and openings that ensure the ventilation of the Rotor Gene Q instrument must not be cover
169. on values increase with fluorescence While this approach makes estimates of nonlinear data more accurate it cannot provide ANOVA as it does not assume a linear model High Resolution Melt analysis High resolution melt HRM analysis characterizes samples based on sequence length GC content and complementarity HRM analysis is used in genotyping applications such as analysis of gene mutations or single nucleotide polymorphisms SNPs and in epigenetics applications for analysis of DNA methylation status HRM analysis provides accurate results and savings on probe and label costs compared to other methods To perform the analysis select Other and then High Resolution Melt Analysis in the Analysis window Double click on the channel to analyze The melt curves from the raw channel are normalized by averaging all starting and ending fluorescence values and then forcing the end points of each sample to be the same as the average 7 64 Rotor Gene Q User Manual 11 2012 Analysis User Interface 7 7 7 7 1 BERotor Gene Q Series Software YIRTUAL MODE high resolution melt demo File Analysis Run Gain View Window Help Settings Progress Profile Temp Samples Analysis Reports Arrange New Open Save Start Pause Stop Help channels 2 Melt 4 FarRed Melt A from 82 FarRed x HRM Normalised Graph Melt A from 82 FarRed Page 1 Normalised Graph Difference Graph
170. on with QIAGEN Kits indicated for use with the Rotor Gene Q instrument is intended for the applications described in the respective QIAGEN Kit handbooks If the Rotor Gene Q instrument is used with kits other than QIAGEN Kits it is the user s responsibility to validate the performance of such product combination for any particular application The Rotor Gene Q instrument is intended for use by professional users such as technicians and physicians trained in molecular biological techniques and the operation of the Rotor Gene Q instrument Rotor Gene Q User Manual 11 2012 2 3 Introduction This page intentionally left blank 2 4 Rotor Gene Q User Manual 11 2012 General Description 3 General Description The Rotor Gene Q is an innovative instrument that enables high precision real time PCR end point PCR and high resolution melt HRM analysis It is highly suited for use in gene expression analysis genotyping pathogen detection and many other areas of research The powerful and user friendly software provides simplicity for beginners as well as an open experimental platform for advanced users Air vents Lid handle Rotor chamber Instrument status lights 3 1 Thermal performance The Rotor Gene Q uses a sophisticated heating and cooling design to achieve optimal reaction conditions The unique rotary format ensures optimal thermal and optical uniformity between samples which is critical for precise and
171. or Gene Q User Manual 11 2012 High Resolution Melt Analysis x 8 H 27 New Open Save As Help The run will take approximately 104 minute s to complete The graph below represents the run to be performed Click on a cycle below to modify it em Insert after ycling HRM Insert before Remove Hold Temperature 95 deg Hold Time 2 mins 0 secs 6 Modify the cycling to suit the amplicon x 72 60 wl New Open Save As Help The run will take approximately 104 minute s to complete The graph below represents the run to be performed UIT Click on a cycle below to modify it Hold Insert after HRM Insert before Remove This cycle repeats _45 time s Click on one of the steps below to modify it or press or to add and remove steps for this cycle 95 deg for 5 secs 10 seconds Acquiring to Cycling on Green inane 60 deg for 10 secs Touchdown Rotor Gene Q User Manual 11 2012 11 13 High Resolution Melt Analysis 7 Ensure fluorescence data will be acquired Acquire data to the green channel at the end of the anneal step Acquisition Same as Previous Mew Acquisition m Acquisition Configuration Available Channel Acquinng Channels To acquire from channel select it from the list in the left and click gt To stop acquiring from channel select it in the right hand list an
172. or Gene Q User Manual 11 2012 Operating Procedures Software Nucleic Acid This is a default template for measuring Concentration nucleic acid concentration using Measurement intercalating dyes HRM This folder contains high resolution melt profiles Other Runs This folder contains additional profiles The cycling and acquisition profiles for all templates can be altered using the wizard Note User defined templates can be added to the template list in the Quick Start wizard by copying or saving ret files to C Program Files Rotor Gene Q Software Templates Quick Start Templates After copying a file to this path the template will appear as an icon in the list If you would like custom icons for your templates create a ico image with the same file name as the template Subfolders can be created to group related templates This allows organization of templates which could be convenient if for example several users are using the same instrument Rotor Gene Q User Manual 11 2012 6 3 Operating Procedures Software 6 1 1 6 4 Rotor selection In the next window select the rotor type from the list Check the Locking Ring Attached checkbox and then click Next B Quick Start 1 Rator Selection 2 Confirm Profile Rotor Type 36 Well Rotor 72 Well Rotor Rotor Disc 72 Rotor Disc 100 M Locking Ring Attached Cancel Rotor Gene Q User Manual 11 2012
173. ore 2 Duo T5500 1 66 GHz or better Main memory 1 GB RAM Hard disk space 10 GB HDD Graphics Adapter and screen with al least 1200 x 800 pixels Interface RS 232 serial port or USB port 4 4 Unpacking the Rotor Gene Q The Rotor Gene Q is delivered with all the necessary components for setting up and running the instrument The box also contains a list of all the components provided Note Check this list for completeness to ensure that all the components are present Note Check that the instrument and delivered accessories are free from transport damage before installation The accessories box sits on top of the foam packing The accessories box contains Installation guide CD software CD user manuals Loading Block 96 x 0 2 ml Tubes Loading Block 72 x 0 1 ml Tubes Rotor Holder dismantled for safe transport 36 Well Rotor this rotor is red in color 36 Well Rotor Locking Ring Rotor Gene Q User Manual 11 2012 4 3 Installation Procedures 4 5 4 6 CAUTION A 4 4 The following items are packed on each side of the foam packing E USB and RS 232 serial cable M International power cable set mM PCR Tubes 0 2 ml 1000 M Strip Tubes and Caps 0 1 ml 1000 Once all these components have been removed from the box remove the foam packing on top of the Rotor Gene Q Carefully remove the Rotor Gene Q from the box and unwrap the plastic cover Open the lid by sliding it towards the back to a
174. ors present in Check that the same master mix was used for all sample samples Too little or degraded template Check the starting quality and quantity of the template 12 2 Rotor Gene Q User Manual 11 2012 Troubleshooting 12 3 Error message Can t open the serial port lt COMPORT gt Chamber Lid Open Could not continue run the chamber lid was opened during a run Please reset the machine and restart the software Chamber Lid Open The instrument chamber lid is open Please close the lid and then click Continue Communication Corrupted General instrument errors Comments and suggestions This error occurs on software startup if the software cannot communicate with the instrument via the configured COM port This is commonly caused by faulty cables loose cables faulty serial ports faulty USB ports a USB driver problem or a USB to serial converter driver problem Reconnect or replace the cable Reinstall the appropriate drivers Start the software in Virtual Mode and select Setup Auto Detect button from the File menu to reset the configured COM port This error occurs when the software has detected the lid is open in the middle of a run Reset the machine and restart the software This error occurs when the user tries to start a run while the instrument lid is open Close the lid of the instrument chamber and then click Continue This error occurs when the data received f
175. otor Gene 0 Series Software 4 7 Build 1 VIRTUAL MODE Current User CORBETT linsongy Administrators Rotor Gene Q User Manual 11 2012 7 61 Analysis User Interface Preparing a run To perform concentration analysis first prepare fluorescent standards and samples ideally in triplicate Preparation of standards standard curve is used to determine the concentration of DNA from each sample measured The DNA used for the standard curve should be a similar type of DNA as in the samples being measured The concentration of at least one DNA sample should be determined using ultraviolet spectrophotometry and this sample should be used as the standard minimum of 3 standards with replicates should be used Importantly DNA standards used in fluorescence detection are only linear within the range of 1 100 ng ul Within this range if the concentration of DNA is halved so is the fluorescent reading The confidence intervals for any concentration outside this range are very broad due to nonlinearity of the chemistry Type of DNA measured Differences have been observed in the measurement of various forms of DNA e g genomic DNA compared to plasmid DNA Therefore only similar DNA types should be measured together and the use of plasmid DNA as a standard should be avoided when measuring genomic DNA Run setup To set up the run select Nucleic Acid Concentration Measurement from the Quick Start wizard 7 6
176. p R 66000 Hun has finished Started at 16 72 2005 3 38 58 AM Finished at 15 12 2005 9 53 40 4M Offset Coefficient 0 Program Version Aotor Gene 6000 Senes Software 1 7 Build 3 Machine Serial No 000620 OF Cancel Apply Machine Options This tab displays settings for the configuration of the Rotor Gene Q Rotor Gene Q User Manual 11 2012 7 67 Analysis User Interface Cancel Apply The rotor should be set to that currently installed in the Rotor Gene Q If opening an existing run this setting will reflect the rotor that was installed in the cycler at that time Messages This tab displays messages indicating if the user has made changes such as pausing the cycler or skipping cycles during a run It also displays warnings received during the run This tab should be checked if results are not as expected Channels If configuring a new run the channels tab displays the current configuration of the available channels If viewing an existing run the information displayed represents the configuration of the channels when the run was performed If a run corrupts the channel settings default channels can be restored by clicking Reset Defaults 7 68 Rotor Gene Q User Manual 11 2012 Analysis User Interface Run Settings General Machine Options Messages Channels Tube Layout Security Create New 10 Green 47 nm Yellow 53 nm Orange X 585nm Hed
177. p down menu on the right hand side of the screen below the toggler 3 The results are automatically calculated and displayed in the Comparative Quantitation Results window below the graph The first columns of the Comparative Quantitation Results window show the sample number and name The Takeoff column gives the takeoff point of the sample The second derivative of the amplification plot produces peaks corresponding to the maximum rate of fluorescence increase in the reaction The takeoff point is defined as the cycle at which the second derivative is at 20 of the maximum level and indicates the end of the noise and the transition to the exponential phase This graph shows a second derivative of an amplification plot showing the relative positions of the second derivative peak and the takeoff point Second derwatwe maximum 40 Takeoff point ARRA The Amplification column provides the efficiency of the sample A 10096 efficient reaction would result in an amplification value of 2 for every sample which means that the amplicon has doubled in every cycle In the raw data the signal should be doubling in the exponential phase For example if the signal was 50 fluorescence units at cycle 12 and then 51 fluorescence units at cycle 13 it should increase Rotor Gene Q User Manual 11 2012 7 45 Analysis User Interface to 53 fluorescence units at cycle 14 All of the amplification values for each sampl
178. paration Sample degradation should be avoided during purification and storage Avoid excessive amounts of inhibitors such as from ethanol carryover To improve HRM results we recommended keeping the amount of template used consistent between samples Spectrophotometric analysis for determining DNA concentration and purity is recommended We recommend QIAGEN kits for sample preparation Note At 260 nm one absorbance unit is equal to 50 ug ml DNA Pure DNA will provide a 260 nm to 280 nm ratio of 1 8 11 7 Software setup 1 Open a new run file by selecting New from the File menu In the Advanced wizard select Rotor Gene Q User Manual 11 2012 11 9 High Resolution Melt Analysis x Quick Start Advanced Corbett Life Science High Resolution Melt run LI HRMT with Pre Amplification Mew Cancel Help M Show This Screen When Software Opens 2 Set the rotor type in this example the 72 Well Rotor is used Ensure that the locking ring is in place and the Locking Ring Attached checkbox is checked before proceeding to the next step 11 10 Rotor Gene Q User Manual 11 2012 High Resolution Melt Analysis x Welcome to the Advanced Aun Wizard Rotor Type 36 Well Rotor Skip Wizard 0 Back gt gt 3 Set the run details Enter the operator name optional and add any notes about the experiment optional Select the reaction volume re
179. peuvent tre g n r es 1 6 Mise au rebut des d chets Les consommables et articles en plastique utilis s peuvent contenir des agents chimiques et infectieux dangereux Ces B 8 Rotor Gene Q User Manual 11 2012 Appendix B d chets doivent tre convenablement collect s et mis au rebut conform ment aux r gles de s curit locales 1 7 Dangers m caniques Quand l appareil Rotor Gene Q est en marche son couvercle doit rester ferm AVERTISSEMENT Pi ces mobiles w13 Afin d viter le contact avec les pi ces en mouvement lorsque l appareil Rotor Gene Q est en marche il convient de laisser son couvercle ferm AVERTISSEMENT Risque d accident corporel et de d t rioration ATTENTION du mat riel W14 Ouvrez et fermez le couvercle du Rotor Gene Q avec A pr caution pour viter de vous coincer les doigts ou les v tements dans l appareil ATTENTION D t rioration de l appareil C5 A V rifiez que le rotor et l anneau de blocage sont correctement install s Si le rotor ou l anneau de blocage pr sentent des signes de d t rioration m canique ou de corrosion n utilisez pas le Rotor Gene Q contactez le support technique de QIAGEN ATTENTION D t rioration de l appareil C6 II ne faut pas utiliser le Rotor Gene Q si le couvercle est bris ou si le verrou du couvercle est endommag V rifiez que le rotor et l anneau de blocage sont correctement install s N utilisez les rotors les annea
180. quired and sample layout desired New Run Wizard This screen displays miscellaneous options for the run Complete the fields clicking Next when you are ready to move to the next page Operator Rotor Gene Operator your mouse over the item for help You Notes Enter any notes for the particular HAM experiment can also click on a help about its available settings Reaction Id Volume pL Sample Layout 1 2 3 Skip wizard lt lt Back Next gt gt Rotor Gene User Manual 11 2012 11 11 High Resolution Melt Analysis 4 Click on the Edit Profile button to modify the times and temperatures of the reaction Hew Run Wizard Temperature Profile This box displays help on elements in the wizard Far help on an item hover Your mouse over the item Far help ou ean also click on a combo bos to display help about its available settings Edit Profile Channel Setup Create New Green 470nm 510rnm Yellow S3 nm 555nm Orange 585nm El nm Red 625nm 6b nm Crimson 680rnm 710hp Remove 4 Onm 5l nm Reset Defaults Gain Optimisation Skip wizard lt lt Back gt gt 5 Set an appropriate initial hold time This time depends on the type of DNA polymerase used The Type it HRM PCR Kit and the EpiTect HRM PCR Kit require a 5 minute activation time The default activation time is 10 minutes 11 12 Rot
181. r Stoffe in Elektro und Elektronikger ten Typenschild auf der Ger ter ckseite Typenschild auf der Ger ter ckseite Typenschild auf der Ger ter ckseite C 12 Rotor Gene Q User Manual 11 2012 Appendix D Appendix D This appendix describes the mathematical techniques used in more detail Quantitation Calculated concentrations are obtained from a simple linear regression model with the known values the log concentrations x and the experimental values the values y The log concentrations and values of the standards are used to construct a model in the form y Mx B Confidence intervals for calculated concentrations We use the following confidence interval 100 1 a for an estimate of a new observation xO from the standard curve Yo 5 rg 1 LS a a a A 2af i H ri This is the confidence interval for the concentration of a single unknown Suppose now we have k further observations at x x0 and we denote their average by Then 3 j a 0 3g 21377270 h and arguments similar to above give Yo jn 1 To 1 I cw r in 2 0 2 H hk n Orr This formula determines how confidence intervals for concentrations of replicate unknowns are determined Rotor Gene Q User Manual 11 2012 D 1 Appendix D For estimation of standards a tighter confidence interval can be obtained Yo 5 1 ro V 1
182. r to decide which channel is appropriate for dye they intend to use The dyes shown in the table are those that are commonly used and do not indicate the limits of the instrument The acquisition options described above also apply to Melt steps except that it is not possible to append acquisition data using the Same as Previous menu Melt and hybridisation A Melt is a ramp between 2 temperatures from a lower to a higher temperature The permitted temperature range is 35 99 C To set up a Melt specify the start temperature the end temperature the temperature increments the length of time to hold at the first acquisition temperature before the ramp is initiated the time each increment is to be held for and the acquisition channels A ramp will be generated between the 2 temperatures If the start temperature is higher than the end temperature the name of the step will change to Hybridisation The Acquiring To option set to Melt A in the screenshot below can be changed by clicking the button The Acquisition window will appear and the channels can be selected Rotor Gene Q User Manual 11 2012 6 17 Operating Procedures Software Ramp from 50 degrees to 90 degrees Rising by degree s each step Wall Far u seconds of pre melt conditioning an first step Wall Far 5 m seconds for each step afterwards Acquire to Melt 4 on Green When running a standard melt the temperature is increased by i
183. re VIRTUAL MODE melt analysis demo EEK File Analysis Gain View Window Help a 7 i OH 9 vw OG OW B o Ww Ww wc Open Save Help Settings Profile Temp Samples Analysis Reports Arrange Channels 7 Cycling A FAM SYER 7 CydngAJOE 2 Melt A CH1 2 Melt A CH2 Ei Melt Curve Analysis Melt A CH1 Page 1 DER 1 2 3 4 5 6 7 8 HE Bank On Bank Of Named On AlOn E dt Samples Peak Calling Flip sign of dF dT Theeshold Temperature Li 785 Current User CORIT1 98 linsonay RG Analyst Rotor Gene Q Series Software 4 7 Buld 1 Current User CORIT1S8 Insongy AG Analyst 6 By logging in as an administrator again RG Operator rights can be assigned to CORIT198 linsongy and the software can be launched again This time the Analysis menu and Reports button are missing and the Run menu is enabled 7 The status bar indicates that the user CORIT198 linsongy belongs to the RG Operator group 7 94 Rotor Gene Q User Manual 11 2012 Analysis User Interface Rotor Gene Q Series Software VIRTUAL MODE melt analysis demo File Run Gain View Window Help 2 OU bb IQ Sw E Open Save Help New Settings Progress Profile F Temperature amp Profile Progress The tun has completed Current User CORIT198 linsongy RG Operator Rotor Gene Q Series Software 4 7 Build 1 VIRTUAL MODE Current User CORIT1S8 Insongy RG Operator
184. re must be installed with the Force Authentication through Windows NT Logon option This queries the Windows domain for your access level and credentials and is essential for providing the accountability and security features Disable simple log in more secure way to log in to Windows is to disable the simple login using the Welcome screen To change the login screen to the classic logon prompt follow these instructions 1 From the Start button select Settings then Control Panel followed by User accounts 2 Select Change the way users log on or off from the dialog box 3 In the next window disable the option Use the Welcome screen and then click Apply Options 101 Back Home Related Tasks Select logon and logoff options Manage accounts Learn About 2 Logon options use Fast User Switching With Fast User Switching you can quickly switch to another user account without having to close any programs Then when the other user is finished you can switch back to your own account Apply Options Cancel Rotor Gene Q User Manual 11 2012 7 85 Analysis User Interface Running as administrator Many users run their computers as an administrator with no password While this is convenient it makes it impossible to determine who is using the computer This eliminates accountability and prevents many Rotor Gene Q software security measures from activating When run
185. ready it is safe to leave it running constantly 2 Insert the Rotor Disc into the Rotor Disc Loading Block using the position one tab on the Rotor Disc and the tube guide holes on the Rotor Disc Loading Block 3 Set up reactions in the Rotor Disc by manual pipetting or using the QIAgility automated liquid handling system Rotor Gene Q User Manual 11 2012 5 9 Operating Procedures Hardware 4 Remove the central portion from one sheet of Rotor Disc Heat Sealing Film by slightly folding the film in halt pinching the center piece and carefully tearing it out 5 Place the film over the Rotor Disc in the correct orientation as shown by the SIDE UP label Ensure that the SIDE UP label is positioned at the bottom of the Rotor Disc Loading Block The central hole in the film should slide easily over the cylinder of the Rotor Disc Loading Block and onto the top of the Rotor Disc 6 Slide the assembly into the Rotor Disc Heat Sealer using the guide rails on the side of the Rotor Disc Loading Block Ensure that the Rotor Disc Loading Block is pushed n completely 5 10 Rotor Gene Q User Manual 11 2012 Operating Procedures Hardware 7 To activate the sealing mechanism first press down on the blue anodized bar at the top of the Heat Sealer then push back the black catch 8 When the sealing mechanism has lowered an orange Sealing light illuminates If the Rotor Disc Loading Block is not
186. reshold 0 30821 Temperature Threshold E Peak Bins Name Value width 4 Bind 8042 4 BinB 8855 4 Mew E dit Remove Imported Settings nane Import Export Rotor Gene Q User Manual 11 2012 7 41 Analysis User Interface 7 42 Flip sign of dF dT Defining peaks Peak Bins Threshold Before defining peaks ensure that the dF dT sign is correct for the data set to give positive peaks In melt curve analysis peaks can be defined and reported using different methods One is to automatically call all the peaks for each sample The other is to assign peaks to bins which is useful for genotyping Bins define the area where peaks are expected to occur The melt curve analysis software clusters peaks into bin groups based on actual peak values in the curve Bins can be edited if required Any peak that is within the defined range of the bin will be assigned to the bin If there are 2 bins close together then the peak will be assigned to the closest bin Note The bins should not be visually positioned to estimate peak positions Set the bins in the approximate area of interest then use the actual reported values in the results table for a more accurate result To define a bin click the New Bin button then click and hold on the graph to define the center of the bin To add another bin repeat the process Use the Remove button to delete bins To
187. rly according to local safety regulations 1 7 Mechanical hazards The lid of the Rotor Gene Q must remain closed during operation of the instrument WARNING Moving parts W13 To avoid contact with moving parts during operation of the Rotor Gene Q the instrument must be operated with the lid closed WARNING Risk of personal injury and material damage W14 CAUTION Open and close the lid of the Rotor Gene Q carefully to avoid trapping fingers or clothing CAUTION Damage to the instrument C5 Make sure that the rotor and locking ring are installed correctly If the rotor or locking ring show signs of mechanical damage or corrosion do not use the Rotor Gene G contact QIAGEN Technical Services Rotor Gene Q User Manual 11 2012 1 7 Safety Information CAUTION Damage to the instrument C The Rotor Gene Q must not be used if the lid is broken or if the lid lock is damaged Make sure that the rotor and locking ring are installed correctly Only use rotors locking rings and consumables designed for use with the Rotor Gene Q Damage caused by use of other consumables will void your warranty CAUTION Damage to the instrument C7 When Rotor Gene Q is started immediately after delivery in cold climates mechanical parts can block Allow the instrument to acclimatize to room temperature for at least an hour before turning the instrument on WARNING Moving parts W15 In case of breakdown ca
188. rom the instrument does not conform to the expected pattern Further investigations are required by a QIAGEN Field Service Specialist to diagnose the problem with the instrument Please contact your distributor or QIAGEN Technical Service Rotor Gene Q User Manual 11 2012 12 3 Troubleshooting Error message Communication Out Sequence Instrument has received data from the machine that is out of sequence Communication Protocol Error communication protocol error occurred with this run Detector motor jam stopped machine Fatal Hardware Malfunction The instrument detected that there was a fatal hardware malfunction Do not attempt to re use the machine until the machine has been serviced by your distributor Comments and suggestions This error occurs when the data received from the instrument are not in the correct order Further investigations are required by a QIAGEN Field Service Specialist to diagnose the problem with the instrument Please contact your distributor or QIAGEN Technical Service This error occurs when the communication protocol configured in the firmware is not the same as the expected protocol Further investigations are required by a QIAGEN Field Service Specialist to diagnose the problem with the communication protocol or the instrument This error can occur when the Rotor Gene Q is started immediately after delivery in cold climates In this case allow the
189. rvice india giagen com Ireland techservice uk giagen com Italy techservice it giagen com Japan techservice jp giagen com Korea South techservice kr qiagen com Luxembourg techservice bnl8qiagen com Mexico techservice mx qiagen com The Netherlands techservice bnl qiagen com Norway techservice nordic giagen com Singapore techservice sgGqiagen com Sweden techservice nordic qiagen com Switzerland techservice ch giagen com UK techservice uk giagen com USA techservice us giagen com 1074319 11 2012 QIAGEN Sample amp Assay Technologies
190. s This opens the Genotyping window which is used to define which genotype is detected in each channel In this window genotypes can be assigned based on the channels in which a sample reacts The channels selected will be used to label the corners of the scatter graph and will guide the user to the general area of the scatter graph in which regions should be defined 4 Genotyping Reacting Channels ld Type Cycling Green Heterozygous Cycling Green Cycling A rellaw Mutant Cycling A rellaw Scatter Graph The scatter graph displays the relative expression of the 2 selected channels The display is normalized to account for different fold increases in each channel and log transformed to accentuate the differences in expression between samples To perform genotyping the user defines regions by clicking and dragging a selection on the graph The selection can then be labeled based on the genotypes configured in the Genotyping window Rotor Gene Q User Manual 11 2012 7 51 Analysis User Interface Scatter Analysis Graph Cycling Green Cycling Yellow Page 1 Scatter graph Scatter graph analysis templates enable analysis genotype and region settings to be templates exported into a single sct file This file can be imported and reapplied in other experiments See Section 8 1 for more details Imported Settings none Import Export 7 6 9 EndPoint analysis EndPoint analy
191. s Two cursors per region are provided defaulted to the ends of the curve The data points within the regions are used to normalize fluorescence the y axis only for the start Region 1 and end Region 2 of the melt plot Data outside the set regions are ignored Adjust the regions to encompass representative baseline data for the premelt and postmelt phases Widening the regions by clicking and dragging allows the software to adjust for the slope of the baseline To ensure curves normalize effectively avoid widening the normalization regions into the melt phase LIT ur 0 ries Senne high mel den Anette Run Gan Ver Wirka Hep ha PHB Hew Open Save 1 J Mele dents Mek alfron 02 FarRed ror Jj Ww amp m Settings Progress Profle Temp Sangles Amys Reports Ama E 1c nalis elt Arom 02 eked Fase 1 in Bis 1 17 Lu ul 0 Sener Sofware 1 7 Dui 94 Note We recommend cursors are only moved if you wish to avoid areas of the melt curve Movement of the cursors toward the melt phase transitions can affect subtraction plots and confidence percentages 3 The Normalised Graph window displays the normalized melt curves Samples can also be viewed as a difference plot against one of the controls 11 20 Rotor Gene Q User Manual 11 2012 High Resolution Melt Analysis aix estere BI hor akoed Fluoresc
192. s in total Al xl File name Three Step with Melt 2008 12 04 1 Save as type Run File rex Cancel 6 6 Rotor Gene Q User Manual 11 2012 Operating Procedures Software 6 1 4 6 2 Sample setup Once the run has started the Edit Samples window allows samples to be defined and described its Edit Samples E 101 xl File Edit Format Security Standard Rotor Style Settings p Conc Format 123457 Samples gt Edit Reset Defaut Gradient E 5 is B ame Type Groups Given Cone Dmm A HR 1000 Standard 1 000 Yes A2 R1000 Standard 1 000 Yes BH n Standard 100 Yes mW A4 R100 Standard 100 Yes 45 R10 Standard 10 Yes AB R10 Standard 10 Yes A R1 Standard 1 Yes Fl Standard Yes BB NTC Yes B2 RNTC NTC Yes EE B3 11000 Standard 1 000 Yes Mss 11000 Standard 1 000 Yes Page ane Page ES E New Delete Synchronize pages Undo OK Cancel Help The Edit Samples window appears after the run has started so that the user can use this time to enter sample names For information about setting up sample definitions in the Edit Samples window see Section 7 8 4 Advanced wizard The Advanced wizard enables options that are not available in the Quick Start wizard such as configuration of gain optimization To use the Advanced wizard select a template by double clicking the template nam
193. s the user to start the run as rapidly as possible The user can select from a set of commonly used templates and enter the minimum of parameters to get started The Quick Start wizard assumes that the reaction volume is 25 ul For other reaction volumes use the Advanced wizard see Section 6 2 As a first step select the desired template for the run by double clicking on the template from the list in the New Run window Rotor Gene Q User Manual 11 2012 6 1 Operating Procedures Software x Quick Start Advanced Imports the cycling and acquisition and sample definitions from the last run open in the software erform Last Run Three Step with Melt ag Two Step lt Quenched FRET Nucleic Acid Concentration Measurement ER Jj al Other Runs Open 4 Template In Another Folder Cancel Help i lv Show This Screen When Software Opens Perform Last Run Perform Last Run uses the cycling acquisition and sample definitions from the last run open in the software Three Step with This is a three step cycling profile and a Melt melt curve with data acquisition on the green channel Two Step This is a two step cycling profile with data acquired on green yellow orange and red channels Quenched FRET This is a three step cycling profile and a melt curve Unlike Three Step with Melt acquisition is at the end of the anneal step 6 2 Rot
194. scence whereas setting below 0 will display samples that decreased in fluorescence during the exponential phase which should clearly be excluded Note If a value is excluded due to activation of either of these techniques a corresponding value in the Quantitation Results window will not be displayed Simultaneously a flag indicating the exclusion will be shown in the Ct Comment column It is therefore important to make sure that the Ct Comment column is displayed at all times In the image below samples 7 8 and 9 were excluded due to the Reaction Efficiency Threshold E Quant Results Cycling A Green Page 1 Bl x Lt Comment Given Cone copies reaction 10e6 Standard MEG 1 00 06 B 1 Standard MEG 1 00 06 9 1 Standard MEG 1 00 06 10 10 5 Standard 15 04 1 00 05 11 10 5 Standard 15 03 1 00 05 12 1065 Standard 15 05 1 00 05 al l Slope amplification reaction efficiency The slope M of a reaction shown in the Standard Curve window can be used to determine the exponential amplification and efficiency of a reaction using the following calculations Exponential amplification 10F1 M Reaction efficiency 100 1 Optimal values for M exponential amplification and reaction efficiency are 3 322 2 and 1 respectively The reaction efficiency is displayed in the report in full and Rotor Gene Q User Manual 11 2012 7 29 An
195. sches Untersuchungsmaterial enthalten sollte immer von einer m glichen Infektionsgefahr ausgegangen werden Wenden Sie nur sichere Laborverfahren an wie sie z B in Ver ffentlichungen wie Biosafety in Microbiological and Biomedical Laboratories HHS www cdc gov od ohs biosfty biosfty htm beschrieben werden Proben Proben k nnen infekti se Erreger enthalten Sie sollten sich der Gesundheitsgetahr bewusst sein die von diesen Erregern ausgeht und derartige Proben gem den erforderlichen Sicherheitsbestimmungen handhaben lagern und entsorgen Rotor Gene Q User Manual 11 2012 Appendix C WARNUNG Proben die infekti se Erreger enthalten w11 Einige Proben die mithilfe dieses Ger ts verarbeitet werden k nnen infekti se Erreger enthalten Gehen Sie beim Umgang mit diesen Proben mit der gr tm glichen Vorsicht und gem den erforderlichen Sicherheits bestimmungen vor Tragen Sie immer eine Schutzbrille zwei Paar Laborhand schuhe und einen Laborkittel Die verantwortliche Person z B der Laborleiter muss alle erforderlichen Vorsichtsma nahmen treffen um sicher zustellen dass die unmittelbare Umgebung des Arbeits platzes sicher ist und die Bediener des Ger ts ausreichend geschult sind Au erdem d rfen die Grenzwerte in Bezug auf infekti se Erreger die in den entsprechenden Sicherheitsdatenbl ttern MSDS oder den Vorschriften der OSHA ACGIH oder COSHH festgelegt sind nicht berschritten werden Be
196. se during operation of the Rotor Gene Q Loose caps and tubes could cause damage to the instrument Rotor Gene Q User Manual 11 2012 13 1 Glossary Term Description Rotor The metal rotor holds tubes or Rotor Discs in the Rotor Gene Q It enables samples to spin in the instrument chamber and ensures that samples are correctly aligned with the optical system The rotor is secured with a Locking Ring Rotor Disc Rotor Discs are circular plates of vertically oriented reaction wells Rotor Disc formats for 72 and 100 reactions are available Rotor Discs are sealed using Rotor Disc Heat Sealing Film and the Rotor Disc Heat Sealer 13 2 Rotor Gene Q User Manual 11 2012 Appendix A Appendix A Technical data QIAGEN reserves the right to change specifications at any time Environmental conditions Operating conditions Power Fuse Heat dissipation thermal load Overvoltage category Air temperature Relative humidity Altitude Place of operation Pollution level Environmental class 100 240 V AC 50 60Hz 560 VA peak Power consumption 60 VA standby Mains supply voltage fluctuations are not to exceed 1096 of the nominal supply voltages F5A 250 V fuse Average 0 183 kW 632 BTU hour Peak 0 458 kW 1578 BTU hour 18 C to 30 C 64 F to 86 F 10 75 noncondensing Up to 2000 m 6500 For indoor use only 2 3K2 IEC 60721 3 3 3M2 IEC 60721 3 3 Rotor Gene Q User Manual 1
197. set the threshold y axis click the icon then click and hold on the graph and drag the threshold line to the desired level Rotor Gene Q User Manual 11 2012 Analysis User Interface Temperature To set a temperature threshold x axis click Threshold the J icon then click and hold on the graph and drag the threshold line to the right This eliminates the threshold line for the lower temperatures Note This is useful when there is noise in the signal at low temperatures Reports This opens the Report Browser where a report can be chosen to preview report can be generated based upon the currently selected channel or a multichannel genotyping report can be generated Results This displays the Melt Curve Results window which shows sample peaks Genotypes Click Genotypes and select the genotypes as shown below 3 Edit Genotypes for Melt Green Genotype Abbrev Bin B Hong JM B NN Heterozygous EE D wild Type KK Lancel Help Es Rotor Gene Q User Manual 11 2012 7 43 Analysis User Interface 7 6 6 This window allows genotypes to be assigned to the incidence of peaks in bins The default genotype configuration is shown in the screenshot with heterozygous samples having 2 peaks homozygous samples a peak in the first bin and wild type samples a peak in the second bin An abbreviation can be typed into the field next to the name of each
198. sions mortelles l int rieur de l appareil Lorsque l appareil est reli l alimentation les bornes peuvent tre sous tension et l ouverture de capots ou le retrait d l ments risque d exposer des l ments sous tension Afin que le Rotor Gene Q fonctionne de mani re satisfaisante et en toute s curit conformez vous aux conseils suivants M Le c ble d alimentation doit tre reli une prise d alimentation disposant d un conducteur de protection terre masse B 4 Rotor Gene Q User Manual 11 2012 Appendix B M Ne r glez ni ne remplacez aucune pi ce interne l appareil M Ne faites pas fonctionner l appareil si des capots ou des pi ces ont t retir s M Si du liquide a t renvers l int rieur de l appareil teignez le et d branchez le de la prise d alimentation puis contactez le support technique de QIAGEN Si l appareil devient dangereux sur le plan lectrique emp chez d autres membres du personnel de l utiliser et contacter le support technique de QIAGEN l appareil peut tre dangereux lectriquement si celui ci ou le c ble d alimentation semble endommag m il a t stock dans des conditions d favorables pendant une longue p riode ila subi des chocs s v res durant le transport AVERTISSEMENT Danger lectrique W9 L appareil pr sente une tiquette de conformit lectrique qui indique le voltage et la fr quence de l alimentation ainsi qu
199. sis B Comparative Quantitation amp Delta Delta CT Relative Quantitation Relative Quant Analysis 8 2 Standard Curves Relative Quantitation Relative Quant Analysis QE Report Delta Delta Ct Condse MIQE Report Delta Delta Ct Full Report Relative Quant Delta Delta TUN Setup The initial setup of the Rotor Gene Q should be completed during installation However this option allows you to change the Rotor Gene Q connection setup if you wish to do so after installation Rotor Gene Q User Manual 11 2012 9 Analysis User Interface xi General Miscellaneous Options The software can simulate basic operation of the machine for testing and traning purposes Ticking this will enable virtual Machine simulation Virtual Mode Untick this bos to prevent Further access to this Setup Screen M Allow access to this setup screen Part COM Auta Detect Virtual Mode Allow access to this setup screen Port Auto Detect Cancel Help Select this option if the software will be used without a connected Rotor Gene Q The software retains all functions This mode is useful for demonstration purposes data analysis and setting up templates If this option is not checked during setup this window can no longer be accessed This security measure prevents users from altering the settings To reestablish access contact your
200. sis allows discrimination between amplified and unamplified samples at the end of a run Results are qualitative positive negative not quantitative 7 52 Rotor Gene Q User Manual 11 2012 Analysis User Interface EndPoint analysis is shown in the screenshot below E Rotor Gene Q Series Software VIRTUAL MODE allelic discrimination demo File Analysis Run Gain View Security Window Help AGM IR OG P O F B Ww AR New Open Save Help Settings Progress Profile Temp Samples Analysis Reports Arrange Channels Cyding A FAM SYBR 27 Cyding A JOE 7 Cyding A from 40 FAM SYBR 27 Cyding A from 40 JOE amp Reports Results Genotypes gt Bank On Bank Off NamedOn All n Al Off Edit Samples Controls Thresholds Positive Controls Heterozygous Positive Control Het Reaction Reaction Heterozygous Unknown Het Reaction Reaction Wild type Unknown Wild Reaction No Reaction Heterozygous Unknown Het Reaction Reaction Heterozygous Unknown Het Reaction Reaction To set the defined controls Wild type Unknown Wild Reaction No Reaction click Edit Samples m um Rotor Gene Q Series Software 4 7 Build 1 VIRTUAL MODE Current User CORBETT linsongy Administrators EndPoint analysis is similar to allelic discrimination in that the results are qualitative and that names can be assigned to certain permutations of reactions over different channels However in EndPoint analysis only a single re
201. sition temperature is compared with the temperature reported by the factory calibration file for the OTV Rotor Disc to verify When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles For more information consult the appropriate material safety data sheets MSDSs available from the product supplier Rotor Gene Q User Manual 11 2012 10 1 Optical Temperature Verification whether the Rotor Gene Q is within temperature specification 10 2 Rotor Disc OTV Kit components The following components are required to run an OTV M Rotor Disc OTV Kit which includes W Sealed Rotor Disc 72 OTV Rotor contains TLCs m Fluorescent scatter plate insert this insert is black for the Rotor Gene 3000 instrument or white for the Rotor Gene 6000 and Rotor Gene Q instruments ACD that contains the following files file with serial number otv OTV test template file ret OTV handbook pdf Certificate of Conformity pdf OTV Reference run rex m Product Sheet M Rotor Gene Series Software Version 1 7 or above which contains the easy to use OTV Rotor wizard Rotor Disc 72 Rotor M Rotor Disc 72 Locking Ring 10 3 Running an OTV 1 Place the fluorescent insert over the emission lens in the bottom of the Rotor Gene Q chamber 2 Place the OTV Rotor Disc into a Rotor Disc 72 Rotor Secure using a Rotor Disc 72 Locking Ring Place the assembly into the Rotor Gene
202. source equivalent Octave This may be useful for methods research Exporting To LinReg LinReg is a tool developed by C Ramakers and coworkers The LinReg tool is available from http LinRegPCR nl Rotor Gene Q software allows the user to export raw data in a format that can then be imported by the LinReg tool for analysis 1 Open the Rotor Gene Q run file containing the raw data Ramakers C Ruijter J M Deprez R H and Moorman 2003 Assumption free analysis of quantitative real time polymerase chain reaction PCR data Neurosci Lett 339 62 8 Rotor Gene Q User Manual 11 2012 Analysis User Interface 7 5 3 7 5 4 2 Export the data to the LinReg export format by selecting Save As and then LinReg Export Format 3 Microsoft Excel automatically displays the exported raw data 4 Start up the LinReg tool The tool asks you to select the cell range where the raw data is located The tool can only analyze one raw channel at a time so an appropriate region of the Excel sheet should be selected Reports After selecting Reports the Report Browser window appears If the data has already been analyzed the report that analysis can be displayed from the Report Browser window Several report types are offered with varying degrees of detail Report Browser loj x Report Categories Templates General Melt Curve Analy
203. ssue Unknown Lung Unknown Lung Unknown Lung Unknown Lung Unknown None None None None None 2 The Edit Group window appears Edit Group X Group Properties Lu Code REAT Mame reated Cancel Help 3 Define a suitable abbreviation then click OK The abbreviation can now be used to set up groups Aggregate results such as average value and 9596 confidence intervals are calculated automatically for groups in any analysis Quant Results Cycling A Green Page 1 E Bl No Name Type Ct Given Conc Cop Cale Conc Copie Var Rep Ct Rep Ct Stc Rep Ct 95 Cl A1 Tissue Unknown 18 82 18 75 0 17 18 48 19 02 A2 Tissue Unknown 18 75 A3 Tissue Unknown 18 92 4 Tissue Unknown 18 52 Lung Unknown 18 73 18 70 0 09 18 55 18 85 AB Lung Unknown 18 62 Lung Unknown 18 81 Lung Unknown 18 63 51 538 Treated Group 18 72 0 13 13 62 18 83 7 82 Rotor Gene Q User Manual 11 2012 Analysis User Interface 7 8 5 Display Options The display options menu is shown below view Window Help Run Settings Temperature Graph Profile Editor Profile Progress Samples vw KL W Settings Progress Profile Temp Samples Gain Calibration Display Options kIw Show at Most 2 Analysis Windows w Show ak Most 6 Windows Reset All Dont Show This Message Again Dialogs Show at Most 2 If this option is checked a ma
204. strument in a safe condition Note Translations in French and German are available in Appendix B and Appendix C The following types of safety information appear throughout this manual WARNING The term WARNING is used to inform you about situations that could result in personal injury to you or other persons Details about these circumstances are given in a box like this one CAUTION The term CAUTION is used to inform you about situations A that could result in damage to the instrument or other equipment Details about these circumstances are given in a box like this one The advice given in this manual is intended to supplement not supersede the normal safety requirements prevailing in the user s country Rotor Gene Q User Manual 11 2012 1 1 Safety Information 1 1 Proper use WARNING Risk of personal injury and material damage w1 CAUTION Improper use of the Rotor Gene Q may cause personal injuries or damage to the instrument The Rotor Gene must only be operated by qualified personnel who have been appropriately trained Servicing of the Rotor Gene Q must only be performed by QIAGEN Field Service Specialists Perform the maintenance as described in Section 9 QIAGEN charges for repairs that are required due to incorrect maintenance WARNING Risk of personal injury and material damage W2 CAUTION Rotor Gene Q is a heavy instrument To avoid personal injury or damage to t
205. t Exit Excel Analysis Sheet LinReg Export Format Matlab Export Save As Use this function to save the run file or data in various formats The options are listed below Run File This saves a copy of the file The user can change the name and the save location This is the default format Template This saves the profile setup and associated settings but not the run data The template can be used to initiate future runs Rotor Gene Q User Manual 11 2012 7 7 Analysis User Interface Run Archive LIMS Export Excel Data Sheet Excel Analysis Sheet LinReg Export Format Matlab Export This saves in a more compact file format Save files in this format before they are e mailed This reduces the time required to send the file and ensures that files are not corrupted by e mail clients This saves the analysis in LIMS compatible formats according to the requirements of the user Please contact QIAGEN Technical Services for more information This exports all the raw channels to an Excel sheet Only the selected samples are exported This exports all the analysis in the current run into a single Excel sheet This exports all raw channel data into a format that can be read by LinReg an efficiency analysis tool See Exporting To LinReg below for more details This exports the data into a format that can be read by the scientific package Matlab or its open
206. t gain levels until it finds one at which the fluorescence levels are i 3 acceptable The range of fluorescence you are looking For depends on the chemistry vau are performing Set temperature to EU degrees Optimize All Optimise Acquiring Perform Optimisation Before 1st Acquisition Perform Optimisation At 60 Degrees At Beginning Of Run Channel Settings Add Min Gain_ Max Gain Edit Green 1 Remove Remove All Set temperature Before reading the Rotor Gene Q will be to Optimise All Optimise Acquiring Perform Optimisation Before 1st Acquisition heated or cooled to match the specified temperature By default this is set as the acquisition temperature Optimise All will attempt to optimize all channels known by the software Optimise Acquiring will only optimize the channels that are used in the thermal profile defined in the run cycling and melt Check this box to perform Gain Optimisation at the first cycle in which data acquisition occurs This is recommended for Auto Gain Optimisation 6 26 Rotor Gene Q User Manual 11 2012 Operating Procedures Software Perform Check this box to perform Gain Optimisation At Optimisation just before starting the run x Degrees At The Rotor Gene Q is heated to the Beginning of specitied temperature Gain Optimisation is Run performed and then cycling begins on the first step usually
207. t it is less likely that the sample amplified than that the negative controls amplified Since this analysis is qualitative such results are not a concern If the negative controls result in higher fluorescence than the positive controls the samples have been incorrectly set up and the following message appears EndPoint Analysis Cycling 4 Green Page 1 Lj E Reports Results Genotypes Graph cannot be displayed as the negative controls are either at the same level or higher than the positive controls Normalization in multiple channels It is possible to analyze signal data over multiple channels but the sample setup is more complex EndPoint analysis assumes that multiplexing is performed and so each tube can only have a single tube position It is not currently possible to analyze a setup in which a sample position is a positive control for one channel and a negative control for another Although only one sample definition per tube position is given in the Edit Samples window normalization occurs independently for each channel It a tube position is a positive control for at least one channel it should be specified as a positive control in the Type column of the Edit Samples window Otherwise its type should be Sample This also applies for negative controls For example if a sample is a positive control in the green channel but not in the yellow channel the sample should Rotor Ge
208. t tee Ede View Took Heb Im Sie Nea DE A B zu SE zm PSE a E lx fe 2 Heterozygous EG 3 Wild ype bx fr 4 Heleozygaous Fz Heterozygous 8 4 Rotor Gene Q User Manual 11 2012 Additional Functions 8 5 Data export To export data in various formats select Data tab in the Export Dialog window The exported file contains the raw data points used in the graph Export Dialog x Picture Native Data Senes Include Paint Index MW Point Labels MW Header Point Colors C HTML Table Excel Delimiter Exporting raw data and analysis data can also be performed by selecting Save As under the File menu see Section 7 5 Spanner wrench icon The spanner wrench icon Li appears at the bottom left of the main window Clicking on the spanner wrench icon enables several options These options can also be accessed by right clicking on the graph Rotor Gene Q User Manual 11 2012 8 5 Additional Functions Adjust Scale Autoscale Revert En Default Scale Export Copy Chart to Clipboard Edit Chart in TeeChart Office Print Digital Filter Select All Named Samples Show Pinpointer Grouping Adjust Scale Autoscale Revert to Default Scale Export Copy Chart to Clipboard Edit Chart in TeeChart Office Print
209. ta No Once the samples are locked it will not be possible to edit samples in the Edit Samples window Rotor Gene Q User Manual 11 2012 7 101 Analysis User Interface 7 9 6 oxi File Edit Format Security Standard Rotor Style Settings Given Conc Format Unit Copies More Options Samples DE alle C IID Name Type Groups Given Conc Selected 1 250bp Unknown Yes 2 2506 Unknown Yes 250 Unknown Yes a 4 500bp Unknown Yes 5 500bp Unknown Yes EN 6 500bp Unknown Yes EN Unknown Yes EN Unknown Yes m 3 Unknown Yes m 10 Unknown Yes 11 Unknown Yes EN 12 Unknown Yes Page pes Page 1 4 gt New Delete Synchronize pages Undo DK Cancel Help Samples can also be locked and unlocked in the Edit Samples window However only an administrator can unlock samples once they have been locked Any unauthorized change to the file will invalidate the Run Signature Locked templates It is currently not possible for the user to create read only template files using the Rotor Gene Q software However if desired it can be specified as a requirement that all runs are performed using a specific template file In order to ensure read only access to this template it should be stored on a network drive where users cannot modify data Users can still run and modify their own profiles while the template on a ne
210. ted fluorescence passes through emission filters on the side of the chamber and is then collected by a photomultiplier The fixed optical path ensures consistent excitation for every sample which means that there is no need to use a passive internal reference dye such as ROX Reaction Chamber Detection Filters m umm PMT Detector Assembly LED Light Source Assembly Tubes in Rotor spin Past Optics Illustration of the optical system Rotor Gene Q User Manual 11 2012 3 3 General Description Available channels Channel Excitation Detection Examples of nm nm fluorophores detected 365 20 460 20 Marina Blue Edans Bothell Blue Alexa Fluor 350 AMCA X ATTO 390 470 10 FAM SYBR Green 1 Fluorescein EvaGreen Alexa Fluor 488 Yellow JOE VIC TET CAL Fluor Gold 540 Yakima Yellow Orange ROX CAL Fluor Red 610 Cy 3 5 Texas Red Alexa Fluor 568 Red Cy5 Quasar 670 LightCycler Red640 Alexa Fluor 633 Crimson 712 high Quasar 705 pass LightCycler Red705 Alexa Fluor 680 High 460 20 5105 SYBR Green resolution SYTO 9 LC Green melt LC Green Plus HRM EvaGreen Note QIAGEN kits indicated for use with the Rotor Gene Q instruments are optimized with respect to certain dye combinations Please refer to the corresponding kit handbooks for example the Rotor Gene Multiplex Handbook or the QuantiTect Virus Handbook for more informati
211. template is selected the wizard guides you through the run setup and allows modification of settings and profiles 11 2012 5 Analysis User Interface xi Quick Start Advanced Imports the cycling erform Last Run and acquisition and s sample definitions f Three Step with Melt from the last run open in the software ag Two Step Fi Quenched FRET LT Nucleic Acid Concentration Measurement FA HRM al Other Runs Open 4 Template In Another Folder Cancel Help i lv Show This Screen When Software Opens For information about the templates provided see Section 6 1 and Section 6 2 New Run New This initiates the run setup using the selected template Cancel This closes this window Help This opens the online help Show This Screen If this box is checked the New Run When Software window is displayed when the software is Opens started 7 6 Rotor Gene Q User Manual 11 2012 Analysis User Interface 7 5 2 Open and Save Open This opens a previously saved Rotor Gene Q run file rex or Rotor Gene Q run archive rea file Open Recent This displays the last 4 files that have been opened or saved Save This saves any changes that have been made to a run file Mew Open Open Recent d Save Import Data Fron Previous Run Template Reports Run Archive LIMS Export Setup Excel Data Shee
212. the unknown sample data points Note If redefining standards to recalculate the standard curve toggling the standard sample visibility to off using the toggler on the right of the screen will remove it from the standard curve calculation Removing standards from the graph to increase the R 2 value is not scientifically valid A failed standard is an indication that the samples may also have failed and so should be included in the results This is the reaction efficiency of the run This value is discussed in more detail on page 7 28 Rotor Gene Q User Manual 11 2012 Analysis User Interface R 2 value The R 2 value or R value is the correlation percentage of the data that is consistent coefficient with the hypothesis that the standards form a standard curve If the R value is low then the standards do not easily fit onto a line of best fit This means that the results i e the calculated concentrations may not be reliable A good R value is approximately 0 99 Note It is possible to achieve a high R 2 value with a poor standard curve if an insufficient number of standards have been run The R 2 value improves as the number of standards decreases For a more accurate indication of the reliability of the results use the confidence intervals on the calculated concentrations as a guide R value square The R value is the square root of the R 2 root of value In general the R 2 value is more correlation
213. their name 7 9 3 Audit trails Every time a file is saved by a user their details are recorded in Run Settings under the Messages tab as Security Audit Trail Summary and Security Audit Trail Detail 7 96 Rotor Gene Q User Manual 11 2012 Analysis User Interface E Security Aue Tied Summary Served Al 26 11 2008 2 47 45 By CORITODT FGQ User 2 Expeirment Slanted 26 11 2008 2 47 46 CORITODI ARGO Use 2 Saved Al 26 11 2008 2 36 00 PM By CORITODTARGQ Use 2 Saved M 26 11 2000 23 12 By CORITOOI ARGO User 2 Secunty Aud Tiol Detal Saved At 26 11 2000 24745 PM Dy CORITOO1 PIGQ User 2 1 5 21 1177220015 1 00 43 0049 4 Lipenment Started 26 11 2008 2 4 45 PH CORIT OO1 6Q User 20515 21117 b Set 7000 2 Saved At 25 13 2008 33600 PN Bp ONTON GO User 2615201 77222 51 E Saved 26 11 2008 3 36 12 PM CORIT ONAGA User 2818 11 Er Che B I Operator skipped end ol cycle lor Cycling Repost 25 She 2415 Deger saring Mek 72 deg 0 Senex Software 1 7 0008 94 MODE Curent User Ure 2 196 Operator This can be used to monitor who has modified the contents of a file The Security Audit Trail Detail contains more detail such as the unique identifier of the user This identifier is important to avoid a user creating an account with the same name on another computer and thereby impersonating another
214. time the threshold is moved in the main window If Fixed the equation does not change because it has been imported from another run Standard Curve conc 10 0 304 CT 11 832 LT 3 292 38 343 Floating Import Curve Reset Import Curve Importing a standard curve allows estimation of concentrations when a standard curve is not available in a particular run and the reaction efficiency has not varied 7 16 Rotor Gene Q User Manual 11 2012 Analysis User Interface between 2 runs Curves can be imported from another channel or from another run by clicking on Import Curve It is possible to adjust the standard curve if required Adiusting the standard curve means that only the efficiency of the source standard curve is imported into the current run Whether or not the standard curve should be adjusted depends on the chemistry used To adjust the standard curve use a reference in the new run with a known concentration Define a reference by setting the sample type to Standard and entering a concentration value in the Edit Samples window Multiple copies of the same reference can be entered to improve accuracy Note that it is not possible to define more than one reference concentration or standard For example it is possible to have 3 replicate references of 1000 copies but it is not possible to have one reference of 1000 copies and another with 100 copies in the same run O
215. ting behavior is characteristic of a particular DNA sample Using HRM the Rotor Gene Q can characterize samples based on sequence length GC content and DNA sequence complementarity HRM can be used in genotyping applications such as analysis of insertions deletions or single nucleotide polymorphisms SNPs or to screen for unknown genetic mutations It can also be used in epigenetics applications for detection and analysis of DNA methylation status It can also be used to quantitatively detect a small proportion of variant DNA in a background of wild type sequence at sensitivities approaching 5 This can be used for example to study somatically acquired mutations or changes in the methylation state of CpG islands HRM on the Rotor Gene Q facilitates multiple applications including Identification of candidate predisposition genes Association studies comparing cases and controls genotype to phenotype Determination of allele prevalence within a population or subgroup SNP screening and validation Screening for loss of heterozygosity DNA fingerprinting Characterization of haplotype blocks DNA methylation analysis DNA mapping Species identification Mutation discovery Determining the ratio of somatic acquired mutations HLA typing HRM is easier and more cost effective than probe based genotyping assays and unlike conventional methods it is a 11 2 Rotor Gene Q User Manual 11 2012 High Resolution Melt Analysis
216. tion analysis Cancel 3 Designate the pages used for normalizing gene analysis and gene of interest analysis For example clicking on Gene of Interest Standard Curve brings up the Selection of Gene of Interest Standard window Select the page where the gene of interest was quantitated Repeat the procedure for the normalizing gene Optionally a calibrator can be defined If this option is selected the calibrator is assigned a value of 1 and all other sample concentrations are calculated relative to this sample Relative Quant Analysis Reports Export Gene Expression 2 Standard Curves Relative Quantitation Gene of Interest Standard Curve Hormaliser Standard Curve Calibrator Defined Rotor Gene Q User Manual 11 2012 7 33 Analysis User Interface Selection of Gene of Interest Standar Currently Selected None To select an existing analysis to use or to create new analysis select a channel from the list then click Select This window will clase and you will be taken to the main window of this analysis 1 Ete Select Other Run Cancel After completing the selections the options will be checked with a check mark as shown below E E Relative Quant Analysis Reports Export Gene Expression 2 Standard Curves Relative Quantitation Gene of Interest Standard Cu Hormal zer Standard Curve Calibrator Define
217. to the 72 Well Rotor by pushing the 3 locating pins through the outer holes of the rotor The Locking Ring ensures that caps remain on tubes during a run Rotor Gene Q User Manual 11 2012 5 7 Operating Procedures Hardware 5 Insert the assembly into the Rotor Gene Q chamber by clicking into place using the locating pin on the rotor hub To remove simply push down on the rotor hub to release and pull out 6 Close the lid and set up the run profile using the Rotor Gene Q software 5 8 Rotor Gene Q User Manual 11 2012 Operating Procedures Hardware 5 3 Rotor Disc setup The Rotor Disc 72 or Rotor Disc 100 comprise 72 or 100 wells respectively in a one piece disc designed for high throughput The Rotor Disc 72 and Rotor Disc 100 do not use caps Instead Rotor Disc Heat Sealing Film is applied to the top and heat sealed using a Rotor Disc Heat Sealer The film prevents contamination by providing a strong durable and tamper proof seal Heat sealing the Rotor Disc is performed as described below IMPORTANT Please read the Product Sheet supplied with the Rotor Disc Heat Sealer before beginning this procedure 1 Switch on the Rotor Disc Heat Sealer using the switch located on the back at the right hand side A red Power light illuminates The Rotor Disc Heat Sealer takes approximately 10 minutes to reach operating temperature when a green Ready light illuminates Note Once the Rotor Disc Heat Sealer is
218. tons at the top right of the graph Edit Profile e New Open Save As Help The run will take approximately 89 minute s to complete The graph below represents the run to be performed Click on a cycle below to modify it Hold Insert after Insert before Remove This cycle repeats 45 time s Click on one of the steps below to modify it or press or to add and remove steps for this cycle Timed Step 95 deg 95 deg for 20 secs 20 seconds Not Acquiring 72 deg for 20 secs Long Range 60 deg for 20 secs Touchdown Long Range Checking this box increases the hold time of the selected step by one second with each new cycle Touchdown Checking this box decreases the temperature by a specified number of degrees for a specified number of initial cycles This is then shown in the display Acquisition Data can be acquired on any channel at any cycling step To set a channel to acquire data click on the Not Acquiring button if a channel has already been set to acquire at this step then the acquiring channels are listed here Rotor Gene Q User Manual 11 2012 6 15 Operating Procedures Software Timed Step 95 10 seconds Mat Acquiring Long Range Touchdown After clicking the Not Acquiring button the Acquisition window appears Same az Previous Mew Acquisition z Acquisition Configuration Available Channels
219. twork drive such as this is protected In order to track which template has been used the Rotor Gene Q software stores the name of the template file that was run This 102 Rotor Gene Q User Manual 11 2012 Analysis User Interface information can be accessed by clicking the Settings button which then enables the Run Settings window to be displayed The template information is stored in Other Run Information Other Aun Fun file has a valid signature Information Template was signed Using Fell carousel hannels saved hor es runc Created with template Dual Labeled Probes ret Hun is currently m progress Started at 16 02 2006 11 16 36 AM 7 10 Gain menu Click on the Gain menu to view the Gain Settings for the current run This sets the gain of the specified channel before a run Gain settings are retained from the last run These can be modified if the run has not yet started or in the initial cycles Use the up down arrows next to each text field to modify the fields Then click OK The gain can be changed during the initial cycles A red line will be drawn in the appropriate channel showing where the gain was changed Cycles before the gain change will be excluded from analysis Green 10 Tz Yellow Orange B Red Crimson rm HAM f Rotor Gene Q User Manual 11 2012 7 103 Analysis User Interface 7 11 7 12 7 12 1 Window menu This menu enab
220. types or tubes from different manufacturers as this will affect optical uniformity We recommend use of tubes from QIAGEN which are specially designed for use with the Rotor Gene Q see Appendix E Tubes from alternative manufacturers may autofluoresce which could affect the reliability of results In addition tubes from alternative manufacturers can vary in length and thickness resulting in misalignment of the optical path of the Rotor Gene Q and the reaction in the tube QIAGEN reserves the right to refuse technical support for problems induced by non QIAGEN certified plastic materials on the Rotor Gene Q instrument IMPORTANT Any use of non QIAGEN certified plastic materials on the Rotor Gene Q may void your instrument warranty IMPORTANT Visually inspect plastic consumables used on the Rotor Gene Q for molding imperfections or inconsistencies Do not use any tubes that are misshapen or flawed CAUTION Damage to the instrument C3 A Visually inspect and make sure the rotor is not damaged or deformed before each run 36 Well Rotor The 36 Well Rotor is red in color The 36 Well Rotor and 36 Well Rotor Locking Ring enable the use of 0 2 ml tubes The Rotor Gene Q User Manual 11 2012 5 1 Operating Procedures Hardware 5 2 tubes do not need to have optically clear caps because the Rotor Gene Q reads fluorescence from the bottom of the tube rather than from the top Domed capped tubes can also be used 7
221. tzt bzw angebracht sind Wenn am Rotor oder Sicherungsring Anzeichen einer mecha nischen Besch digung oder von Korrosion zu erkennen sind verwenden Sie den Rotor Gene Q nicht mehr kontaktieren Sie den Technischen Service von QIAGEN ACHTUNG Ger tesch den C6 Der Rotor Gene Q darf nicht benutzt werden wenn der Deckel oder die Deckelverriegelung besch digt ist Stellen Sie sicher dass der Rotor und der Sicherungsring ordnungsgem eingesetzt bzw angebracht sind Verwenden Sie ausschlie lich Rotoren Sicherungsringe und Verbrauchsartikel die f r die Verwendung mit dem Rotor Gene Q vorgesehen sind F r Sch den die durch die Verwendung anderer Verbrauchsartikel verursacht werden wird keine Garantie bernommen Rotor Gene Q User Manual 11 2012 C 9 Appendix C ACHTUNG Ger tesch den C7 Wenn der Rotor Gene Q sofort nach Lieferung unter kalten Klimabedingungen in Betrieb genommen wird k nnen mechanische Teile eventuell blockieren Lassen Sie das Ger t zuerst f r mindestens eine Stunde bei Raumtemperatur stehen bevor Sie es einschalten WARNUNG Sich bewegende Ger teteile W15 Im Falle einer St rung die durch einen Stromausfall verursacht ist ziehen Sie das Stromkabel aus der Steck dose und warten Sie 10 Minuten bevor Sie versuchen den Deckel manuell zu ffnen WARNUNG berhitzungsgefahr Wi Vergewissern Sie sich dass ein Mindestabstand von 10 cm zwischen Seitenw nden bzw
222. u fabricant risque de compromettre la protection offerte par l appareil AVERTISSEMENT Risque d accident corporel et de d t rioration ATTENTION du mat riel 7 La pr sence de papier sous le Rotor Gene emp che l appareil de refroidir Il est recommand de lib rer l espace sous l appareil Rotor Gene Q User Manual 11 2012 B 3 Appendix B ATTENTION D t rioration de l appareil C2 Utilisez toujours un anneau de blocage sur le rotor Cela emp che les bouchons de sortir des tubes pendant l essai Si des bouchons sortent pendant un essai ils peuvent endommager la chambre ATTENTION D t rioration de l appareil C3 Veuillez inspecter l instrument et vous assurer que le rotor n est pas endommag ni d form avant chaque cycle Si vous touchez le Rotor Gene Q pendant l essai alors que vous tes charge en lectricit statique dans de graves cas le Rotor Gene Q peut se r initialiser Toutefois le logiciel red marre alors le Rotor Gene Q et poursuit l essai 1 2 S curit lectrique Avant l entretien debranchez le cordon d alimentation de la prise de courant AVERTISSEMENT Danger lectrique w8 Toute interruption du conducteur de protection conducteur de terre de masse l int rieur ou l ext rieur de l appareil ou toute d connexion de la borne du conducteur de protection est susceptible de rendre l appareil dangereux Toute interruption intentionnelle est interdite Ten
223. ures 9 Maintenance Procedures Maintaining the working performance of the Rotor Gene Q is easy Optical performance is maintained by ensuring that the lenses located at both the emission and detection source are clean This is achieved by gently wiping a cotton tip applicator moistened with ethanol over the lenses Note Clean the lenses at least once a month depending on usage Wipe the rotor chamber at the same time Keep the work bench area clean and free from dust and sheets of paper The air inlet of the Rotor Gene Q is at the bottom and loose material such as paper or dust may compromise performance To avoid dust build up keep the lid of the Rotor Gene Q closed when the instrument is not in use If the rotor chamber becomes contaminated it can be cleaned by wiping the surfaces with a lint free cloth dampened but not dripping with a 0 196 v v bleach solution Wipe the chamber with a lint free cloth dampened with PCR grade water to remove traces of bleach When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles For more information consult the appropriate material safety data sheets MSDSs available from the product supplier Rotor Gene Q User Manual 11 2012 9 Maintenance Procedures This page intentionally left blank 9 2 Rotor Gene Q User Manual 11 2012 Optical Temperature Verification 10 Optical Temperature Verification Optical Temp
224. used by power failure remove the power cord and wait 10 minutes before attempting to manually open the lid WARNING Risk of overheating To ensure proper ventilation maintain a minimum clearance of 10 cm at the sides and rear of the Rotor Gene Q Slits and openings that ensure the ventilation of the Rotor Gene Q must not be covered 1 8 Heat hazard WARNING Hot surface W17 The Rotor Gene Q chamber can reach temperatures above 120 C 248 F Avoid touching it when it is hot 1 8 Rotor Gene Q User Manual 11 2012 Safety Information WARNING Hot surface W18 When a run is paused the Rotor Gene Q will not be cooled completely to room temperature Exercise caution before handling the rotor or any tubes in the instrument 1 9 Maintenance Perform the maintenance as described in Section 9 QIAGEN charges for repairs that are required due to incorrect maintenance WARNING Risk of personal injury and material damage W19 CAUTION Only perform maintenance that is specifically described in this user manual WARNING Risk of fire W20 When cleaning the Rotor Gene Q with alcohol based disinfectant leave the Rotor Gene Q lid open to allow flammable vapors to disperse Only clean the Rotor Gene Q when the chamber has cooled down WARNING Risk of electrical shock CAUTION Do not disassemble the Rotor Gene Q instrument A CAUTION Damage to the instrument housing C8 Never clean th
225. user In this case the user names will be the same but the account IDs will be different Rotor Gene Q User Manual 11 2012 7 97 Analysis User Interface 7 9 4 The identifier for the account CORITOO1 RGQ User 2 S 1 5 21 1177238915 195 is shown in the details 2H PH jw 5 4 wD Hew Open et Fours Stop Hep Settings Progress Profle Temp Samples furange J Secun Trad Summam Sarees Ab 2EVTY AUC 2 37 45 PH COA TOOT 91050 Us 2 Eigener Slate Al 25 71 1 2008 2 47 85 PM By AG User 2 Sarre 1 72008 3 35000 COTTON ROO Ups 2 G r EU QC 3 265 12 COTTON ARE Less 2 Send EU E208 41 58 CORITDOS AR Less 2 5 Diniy dui Dead Sarew fu 25071152008 2 47 40 CORITDOS AU Uis 2 5 145 71 0 17723883101 5 15 Eigener Slatted fu Fe 172008 7 47 PM By CORT OOD Uter 2 51459214117 f dni CT OO 776 00 a COC Ti 2 5 845 21 1177 238500 fied OO EI CORE TOR AGO 2 5 135 271 1172 20888511 E 15 E Operator hied end al cycle lox Cucing Piepaat 25 0 Sene Software 1 7 Dube S4 va TUAE MODE Curent User CORITTOTI IS Ure 2 1 Run Signatures The audit trail is stored in the Rotor Gene Q run file To avoid any unwanted modification of these files they should be kept in a safe location accessible only by designated Windows accounts However if files are stored
226. ux de blocage et les consommables con u cet effet qu avec le Rotor Gene Q La d t rioration due l utilisation d autres consommables annulera votre garantie Rotor Gene Q User Manual 11 2012 B 9 Appendix B ATTENTION D t rioration de l appareil C7 Si le Rotor Gene Q est allum juste apr s sa livraison par des temp ratures basses les pi ces m caniques peuvent se bloquer Laissez l appareil arriver temp rature ambiante pendant au moins une heure avant de l allumer AVERTISSEMENT Pi ces mobiles W15 A En cas de panne provoqu e par une coupure de courant d branchez le cordon d alimentation et attendez 10 minutes avant d essayer d ouvrir manuellement le couvercle AVERTISSEMENT Risque de surchauffe w16 Afin de garantir une bonne ventilation laissez un d gagement d au moins 10 cm sur les c t s et l arri re de l appareil Rotor Gene Les fentes et les ouvertures qui garantissent la ventilation du Rotor Gene Q ne doivent pas tre obstru es 1 8 Danger li la chaleur AVERTISSEMENT Surface chaude W17 La chambre du Rotor Gene Q peut atteindre des temp ratures sup rieures 120 C 248 F Evitez de le toucher lorsqu il est chaud AVERTISSEMENT Surface chaude W18 Pendant la pause d un cycle le Rotor Gene Q ne revient pas compl tement temp rature ambiante Faites attention avant de manipuler le rotor ou de quelconques tubes dans l appareil
227. ware durchgef hrt und der Rotor Gene Q setzt das Experiment fort 1 2 Schutz vor Stromschlag Ziehen Sie das Netzanschlusskabel aus der Steckdose bevor Sie Wartungsarbeiten am Ger t vornehmen WARNUNG Gefahr durch Stromschlag w8 Jede Unterbrechung des Schutzleiters Erdungs bzw Masseleiter im Ger t oder au erhalb des Ger ts und jede Abtrennung des Schutzleiters am Anschluss der Netzleitung erh ht die Gefahr eines Stromschlags Eine absichtliche Unterbrechung der Schutzleiterverbin dung ist verboten Gef hrliche Spannung im Ger t Wenn das Ger t an die Stromversorgung angeschlossen ist sind die Anschlussstellen spannungsf hrend Durch das ffnen der Abdeckungen oder das Entfernen von Geh useteilen k nnen spannungsf hrende Komponenten freigelegt werden C 4 Rotor Gene Q User Manual 11 2012 Appendix C Um einen zufriedenstellenden und sicheren Betrieb des Rotor Gene Q zu gew hrleisten befolgen Sie bitte die nachstehenden Hinweise M Das Netzkabel muss an eine Wechselstrom Steckdose mit Schutzleiter Erdungs Masseleiter angeschlossen werden M Nehmen Sie im Ger teinneren keine Einstellungen an Teilen vor und wechseln Sie keine Teile aus E Nehmen Sie das Ger t nicht in Betrieb wenn Abdeckungen oder Teile entfernt worden sind M Falls Fl ssigkeit auf dem Ger t versch ttet wird und in das Ger t l uft dann schalten Sie es sofort aus trennen Sie es von der Netzspannung Stecker ziehen und s
228. ximum of 2 Analysis analysis windows are shown at once If Windows multiple windows are opened readability may be affected Checking this option closes the first analysis window and replaces it with the last opened window It the option is unchecked more than 2 analysis windows can be displayed Show at Most 6 To improve readability the software Windows removes unused windows when new windows are opened This option is enabled by default as it keeps the Rotor Gene Q software screen clear If it is necessary to see more than 6 windows at once uncheck this option Reset All Don t If this is selected the software will redisplay Show This all dialog boxes where the Do not display Message Again this message again checkbox was Dialogs checked These include messages about suspicious settings that may have previously been set not to display again This may be useful for a new user who is unfamiliar with the Rotor Gene Q or the Rotor Gene Q software Rotor Gene Q User Manual 11 2012 7 83 Analysis User Interface 7 9 Security menu The Rotor Gene Q software includes features that enable it to operate securely When correctly configured the Rotor Gene Q software can ensure the following Access to the Rotor Gene Q or the analysis software is restricted to user groups Modifications to run files are logged Unauthorized modifications are detected signatures Templates used to perform runs are logged S
229. y 4 Switch on the Rotor Gene Q by moving the toggle switch located at the back on the right hand side to the position A blue Standby light on the front of the Rotor Gene Q indicates that the instrument is ready for use Note When starting connected to a computer for the first time the Rotor Gene Q will be recognized by the operating system and a number of messages will appear Please refer to the Rotor Gene Q Installation Guide provided with the instrument CD and printed edition for guidance 5 Double click the Rotor Gene Q Series Software desktop icon to initiate the software Software Rotor Gene Q User Manual 11 2012 4 7 Installation Procedures Welcome window appears the first time the software is started but does not appear for subsequent software upgrades Introductory Screen and Initial Setup Welcome Welcome Before vou begin the analysis software needs to know a couple of things about the type of system you are using Machine Serial Number Part COM Auto Detect Run in Virtual Mode For Demonstration 1 Begin Exit Program Machine Serial Type in the serial number 7 digits which Number can be found on the back of the Rotor Gene Port Choose either USB or serial cable Select the appropriate communications port or click the Auto Detect button Auto Detect When using this option the corresponding USB or serial port wi
230. y This is expressed in scientific notation Note The value displayed in Average Amplification to the right of the represents the standard deviation of the average amplification after removal of outlier amplification 7 46 Rotor Gene Q User Manual 11 2012 Analysis User Interface values If this value is large then there may be a large error in the overall calculated concentration values Relative concentrations are calculated by the software as follows 1 The takeoff point of each sample is calculated by looking at the second derivative peaks 2 The average increase in raw data 4 cycles after the takeoff is calculated This is the amplification value for the sample 3 Outlier amplifications are removed to account for noise in background fluorescence 4 The remaining amplifications are averaged This is the average amplification 5 The average takeoff point is calculated for each calibrator replicate 6 The relative concentration for a sample is calculated as Amplification Calibrator takeoff Sample takeoff 7 The result is displayed in scientific notation in the Rep Conc column of the Comparative Quantitation Results window 7 6 7 Allelic discrimination Allelic discrimination uses real time kinetic data from 2 or more channels to genotype samples To perform this analysis select Other and then Allelic Discrimination in the Analysis window When performing allelic discrimination
231. ysis HRM analysis enables both visual and auto calling of genotypes Results can be viewed as either a normalized melt plot or a difference plot Normalized curves provide the basic representation of the different genotypes based on curve shifting for homozygotes and curve shape change for heterozygotes Difference plots are an aid to visual interpretation They plot the difference in fluorescence of a sample to a selected control at each temperature transition Difference plots provide an alternative view of the differences between melt curve transitions Note First derivative melt curve analysis as used by the standard Melt option in the Analysis window is considered inappropriate for HRM analysis This is because any derivation of the data adds artificial noise and makes data interpretation more difficult The following steps describe the analysis of HRM results using Rotor Gene Q software 1 Select the option from the Analysis window Analysis 7 7 x 2 Std Curves Rel Other Quantitation Melt w HAM A HRM Page 1 Rotor Gene Q User Manual 11 2012 11 19 High Resolution Melt Analysis 2 Windows appear showing the raw data the normalized graph and the results The raw data window allows adjustment of the regions of normalization Normalization allows all the curves to be compared with the same starting and ending fluorescent signal level to aid interpretation and analysi

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Rotor-Gene® Q User Manual - Frank`s Hospital Workshop