Home

OpenArray® Real-Time PCR System, Digital PCR Experiments

1. Sample Dilution Assay Load to wells Sample 1 2 0 Assay 1 Al A2 B1 B2 Sample 1 1 0 Assay 1 A3 A4 B3 B4 Sample 1 0 5 Assay 1 Ab A6 B5 B6 Sample 2 2 0 Assay 1 A7 A8 B7 B8 Sample 2 1 0 Assay 1 A9 A10 B9 B10 Sample 2 0 5 Assay 1 A11 A12 B11 B12 Sample 1 2 0 Assay 1 C1 C2 D1 D2 Sample 1 1 0 Assay 1 C3 C4 D3 D4 Sample 1 0 5 Assay 1 C5 C6 D5 D Sample 2 2 0 Assay 1 C7 C8 D7 D8 Sample 2 1 0 Assay 1 C9 C10 D9 D10 Sample 2 0 5 Assay 1 C11 C12 D11 D12 Sample plate Plate area 1 Plate area 2 Yate area 4 eae q 1 te area 6 SSS 1 area 8 quee Y 4 Subarray A1 Te ESI ESTERI EIER E IERI EESTI t9 E E ES E cd ES E e E US E EE EE ES EE Ee EE d d Ee EE SET ES EE ESSERE 9 OpenArray plate SS Subarray D12 When you transfer the samples from the sample plate to the OpenArray Digital PCR Plate program the OpenArray Accufill System or AutoLoader System to perform one load The system transfers the samples to the following locations of the OpenArray Digital PCR Plate Sample plate Load OpenArray Digital PCR Plate subarray locations 1 1 Through holes A1 through H8 Applied Biosystems OpenArray9 Real Time PCR System Digital PCR User Guide 55 56 Appendix B Example Layouts Example plate layout 2 Applied Biosystems OpenArray9 Real Time PCR System Digital PCR User Guide APPENDIX C Backgroun
2. nann cece eect n 51 Example plate layout 1 2 2 2 eet I eeeeeees 52 Example plate layout 2 0 0 2 eet n 54 Background Information ele 57 PCR and the 5 nuclease assay 0 0 cece cece cette nn 58 Threshold cycle ICs data calculation uror ders ee Eee RD leh b acted 59 Quantitative analysis and copy number calculation 0 00 eee eee eee eee 60 Sally ossnic4aP RE ROSAS EE RRaLEIMRCEE ESI CER RE 61 Symbols on instruments sneins tedd 00 ebi dak dA iae a nn 62 General instrument safety 0 00 nunne nrnna arenneren 63 Physicalhazard safely ib e ERST Ene eate esa A 64 Electrical safety eet pux s e RUE iau Ree E vd pu tud 64 Workstation safety 00 0 aa eect a E A d eE e eee eee 65 Safety and electromagnetic compatibility standards 0 00 000 e cece 66 General chemical safety 0 00 c cece cece eee eee nn 67 SDSS ests a Aine eet ane ee alee aes are LA o A MALE Ae 68 Chemical waste safety annann cece cece ee eee eee nn 69 Biological hazard safety ise ea ead RARI Sees WG RES ME 70 Chemical alertss 2 8 eset iC eto utet s 70 Bibliography 5c exsecutio EP aoieanna MED EE eee 71 GlOSSary wei ii keemia rada i rn n renei ara 73 Applied Biosystems OpenArray Real Time PCR System Digital PCR User Guide About This Guide Purpose The Applied Biosystems OpenArray Real Time PCR System Digital PCR User Guide provides inform
3. 000 e eee eee Materials insist headit ced dan bet eee Workflow x J 5 n gabe e e rta e RIA CHAPTER 1 Overview 1 Chapter 1 Overview Introduction Introduction OpenArray Real Time PCR System The OpenArray Real Time PCR System can be used to perform and analyze digital PCR experiments for accurate and sensitive quantitation of nucleic acid targets The following components are required to perform digital PCR on the OpenArray System e OpenArray Real Time PCR System Instrumentation used to load digital PCR samples that performs thermal cycling and quantitative detection of targets using real time analysis see below e OpenArray Digital PCR Plates Reaction vessels used to load and contain the digital PCR reactions for thermal cycling and the subsequent imaging by the OpenArray Real Time PCR System see page 9 for more information TaqMan OpenArray Digital PCR Master Mix and Assays Fluorescence based polymerase chain reaction PCR reagents used to amplify and detect nucleic acid targets for digital PCR analysis see page 10 for more information e OpenArray Digital PCR Software Software used to perform a copy number and Poisson statistical analysis of the digital PCR experiments performed on the OpenArray Real Time PCR System see page 11 for more information The OpenArray System consists of the following components e OpenArray AccuFill System OpenArray AutoLoader Loads your sa
4. Template or amplicon contamination Follow established PCR good laboratory practices Applied Biosystems OpenArray9 Real Time PCR System Digital PCR User Guide 43 Chapter 4 Troubleshooting Observation Possible cause Recommended solution Amplification curve shows weak amplification Sequence mismatches between target and assay sequences Perform bioinformatics For more information refer to the e Custom TaqMan Genomics Assays Protocol Submission Guidelines PN 4367671 e Bioinformatic Evaluation of a Sequence for Custom TaqMan Gene Expression Assays Tutorial from www appliedbiosystems com Degraded reagents and or probe e Check the expiration date of the reagents e Verify that you followed the correct handling and storage conditions e Avoid excessive freeze thaw cycles Consider diluting the 60X TagMan assay to a 20X working stock Degraded or contaminated template Improve the sample integrity extraction methods See Prepare the DNA samples on page 18 e Check each template preparation by agarose gel electrophoresis or bioanalyzer to determine the Purity only one product should be formed Level of degradation e Use RNase free sterile filtered water Inhibitors present in the reaction Verify the presence of an inhibitor a Create a serial dilution of your sample b Run the serial dilution with an expressing assay for example a
5. Running the sample using an alternative assay for the same gene e Verify the known expression of the gene in the sample type Note If the recommended actions do not resolve the problem the result may be correct The reaction may not have enough copies of the target gene Sample is too dilute Verify by e Rerunning the sample using the same assay e Rerunning the assay using more sample e Running the sample using an alternative assay for the same gene Note If the recommended actions do not resolve the problem the result may be correct Perform a dilution series of the sample by increasing the quantity of input DNA added to the first point of the series Decrease in ROX dye fluorescence passive reference dye Precipitation in the TaqMan buffers When using the TagMan PCR Core Reagents Kit be sure to mix the tubes well e Use TaqMan OpenArray Digital PCR Master Mix Be sure to mix thoroughly to produce a homogenous solution Degraded TaqMan buffers Verify that the kits have been stored according to the instructions on the packaging and have not expired Simultaneous increase in fluorescence from both the e Passive reference ROX dye e Reporter dye s Evaporation Check the seal of the optical adhesive cover for leaks No template control NTC shows amplification Contaminated reagents contaminated with gDNA amplicon or plasmid clones
6. No right to perform LTC s patented Digital PCR Methods is conveyed with the purchase of this product A license to perform LTC s patented Digital PCR Methods that employ devices with multiple sample chambers can be obtained with either i purchase of an Authorized Digital PCR Array or ii a separate license from Life Technologies For licensing information please contact outlicensing dlifetech com or Out Licensing Life Technologies 5791 Van Allen Way Carlsbad California 92008 TRADEMARKS The trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners TaqMan is a registered trademark of Roche Molecular Systems Inc Apple Safari and Macintosh are trademarks of Apple Inc Microsoft and Internet Explorer are trademarks of Microsoft Corporation Mozilla is a trademark of Mozilla Foundation LabView is a registered trademark of National Instruments Corporation 2010 Life Technologies Corporation Corporation All rights reserved Part Number 4459761 Rev A 10 2010 Contents About This Guide reet ERR 5 npe 5 AuUdier68 7 2 56 Lent ntt inet atii aet DP oris tet Anar of 5 AsSUmbplioris i esce etre retta teed a natal hal E d ura Oral ae ona 5 Safety information xo erbe dun ce Satine Deb ase qug e ee Bae s 6 CHAPTER 1 OVEIVICW AMT et 7 MEFO AUCTION a bot a Reb rre hil gin eee e Pr EUER e piii t ctfi 8 Installthe SoftWare J etes rere ae che eda Aedes 12 Materials so EI
7. Rerun the assay using new reagents Besure your workspace and equipment are cleaned properly Use AmpErase UNG e Run no reverse transcription controls to rule out genomic DNA contamination e gDNA contamination only Design an assay that spans an exon exon boundary 46 Applied Biosystems OpenArray9 Real Time PCR System Digital PCR User Guide General guidelines APPENDIX A Prevent Contamination PCR assays require special laboratory practices to avoid false positive amplifications The high throughput and repetition of these assays can lead to amplification of one DNA molecule Note Aftera OpenArray Digital PCR Plate has been sealed in a TaqMan OpenArray Case it is less likely to spread contamination than other types of reaction plates PCR good laboratory practices When preparing samples for PCR amplification Wear a clean lab coat not previously worn while handling amplified PCR products or used during sample preparation and clean gloves Change gloves whenever you suspect that they are contaminated Maintain separate areas and dedicated equipment and supplies for Sample preparation PCR setup PCR amplification Analysis of PCR products Never bring amplified PCR products into the PCR setup area Open and close all sample tubes carefully Try not to splash or spray PCR samples Keep reactions and components capped as much as possible Use a positive displacement pipette or aerosol
8. Sample Group Displays the analyzed data grouped by sample 2 Export Results Saves the analyzed data to a x Microsoft Excel spreadsheet xls file 3 Results table Displays the results of the analysis where each row contains the following analyzed data for a sample or replicate group Group The name of the sample or replicate group Average Copies Per Reaction The number of copies calculated for the associated sample or replicate group Lower Upper Confidence Level The lower and upper confidence intervals calculated for the associated sample or replicate group Note See Change the analysis settings on page 39 to change the confidence interval used by the OpenArray9 Digital PCR Software Total Omitted The total number of through hole reactions for the sample or replicate group that the software omitted from the analysis Note See About outliers on page 36 for more information regarding the identification and removal of outliers Confidence Interval Range The lower and upper confidence intervals calculated for the associated sample or replicate group displayed as a hyphenated range 32 Applied Biosystems OpenArray9 Real Time PCR System Digital PCR User Guide About the Results plot Bar Plot of Copies Per Reaction 20 Chapter 3 Analyze the Results View the results Average Copies Per Reaction 4 Bar Plot of Copies Per Reaction Displays the calculated number
9. e Dilutions of two samples S01 and S02 are evaluated for one nucleic acid target using a single TaqMan assay A01 Aten fold six point dilution series of each sample is evaluated for the nucleic acid target The dilutions are such that each through hole reaction contains either 1 0e4 1 0e3 100 10 1 or 0 1 dilution of the stock solution The samples are arrayed in replicate so that the plate evaluates each sample dilution assay combination in quadruplicate The plate contains a total of 12 replicate groups where each group consists of 256 replicate reactions 4 subarrays x 64 through holes 256 reactions Experiment layout example experiment The following figure illustrates the OpenArray 384 well sample plate layout for the 1 2 3 4 5 6 S01 S01 S01 S01 100 10 1 0 1 A01 A01 A01 A01 S01 S01 S01 S01 100 10 1 0 1 A01 A01 A01 A01 S01 S01 S01 S01 100 10 1 0 1 A01 A01 A01 A01 S01 S01 S01 S01 100 10 1 0 1 A01 A01 A01 A01 9 10 11 12 S02 S02 S02 S02 100 10 1 0 1 A01 A01 A01 A01 S02 S02 S02 S02 100 10 1 0 1 A01 A01 A01 A01 S02 S02 S02 S02 100 10 1 0 1 A01 A01 A01 A01 S02 S02 S02 S02 100 10 1 0 1 A01 A01 A01 A01 52 Applied Biosystems OpenArray9 Real Time PCR System Digital PCR User Guide Sample plate setup Subarray locations Appendix B Example La
10. 13 Work TOW ears enaa a Mok e Ea Ping eh pate A d NER aaa ea 15 CHAPTER 2 Prepare and Perform the Digital PCR Experiments 17 Prepare the DNA samples 00 cece eee n 18 Prepare the digital PCR experiment 000 cece cee ee 20 Performthe ncididunt an uc sich eee dee n detto ade 23 CHAPTER 3 Analyze the Results 1 25 21i IIR oats ened ivehens 27 Oper the experiment 22 2 eos cece et edges ae AONA dew bbe eG eee EES MIR 28 Edit the experiment information 000ce cece cece e 30 Viewsthe results ia coe hb tre cathe ia reru erar ebrei us 32 Viewhedala n d eee nd deese i aa e ah ede O ec tna Mladen tes 34 Review and omitoutliers 2 0 00 cee eee hn 36 Optimize the analysis settings 0 0 00 c cece cece cette eeee 38 Save and export the results 0 200 cece eee eee cece eet e 39 CHAPTER 4 Troubleshooting strisa oeira dra aaeh RR eiu aE IEEE 41 Applied Biosystems OpenArray Real Time PCR System Digital PCR User Guide 3 Contents APPENDIX A APPENDIX B APPENDIX C APPENDIX D Prevent Contamination eese 47 General guidelines msiri iae a a a T Aa a n 47 PCR good laboratory practices 00 0c cece cect ee 47 Clean the OpenArray Autoloader accessories 0 0000 cece eee eee cence nee 47 Example Layouts 1 5 2522 ieisuc ii gie gd ur REF EVE 49 About the OpenArray plates 2 0 0 0 ccc ccc cnn nen eee n 50 Digital PCR experiment setup
11. Each 4 liter bottle should be secured in a low density polyethylene safety container with the cover fastened and the handles locked in the upright position Wear appropriate eyewear clothing and gloves when handling reagent and waste bottles i WARNING CHEMICAL STORAGE HAZARD Never collect or store waste in a glass container because of the risk of breaking or shattering Reagent and waste bottles can crack and leak Each waste bottle should be secured in a low density polyethylene safety container with the cover fastened and the handles locked in the upright position Wear appropriate eyewear clothing and gloves when handling reagent and waste bottles Chemical safety To minimize the hazards of chemicals guidelines Read and understand the Safety Data Sheets SDSs provided by the chemical manufacturer before you store handle or work with any chemicals or hazardous materials See About SDSs on page 68 Minimize contact with chemicals Wear appropriate personal protective equipment when handling chemicals for example safety glasses gloves or protective clothing For additional safety guidelines consult the SDS Minimize the inhalation of chemicals Do not leave chemical containers open Use only with adequate ventilation for example fume hood For additional safety guidelines consult the SDS Check regularly for chemical leaks or spills If a leak or spill occurs follow the manufacturer s cleanup procedu
12. OpenArray Real Time PCR System 4408510 TaqMan OpenArray Digital PCR Master Mix 2X 1 5mL 4458086 _ PPlied Biosystems 5 0 mL 4458080 Optional Fine tip marker MLS Table 2 OpenArray Digital PCR Plate sealing Product Part Source number TaqMan OpenArray Accessories Kit 4453975 e TagMan OpenArray Case e OpenArray Sealing Glue Applied e OpenArray Immersion Fluid Biosystems OpenArray Case Sealing Station 4409361 25 Slide Holder 4407056 Ethanol MLS Razor blade MLS Table 3 General use Product part Source number Non Stick RNase free Microfuge Tubes 0 5 mL 500 tubes AM12350 Non Stick RNase free Microfuge Tubes 1 5 mL 250 tubes AM12450 _ Applied Biosystems Non Stick RNase free Microfuge Tubes 2 0 mL 250 tubes AM12475 Centrifuge with plate adaptor Disposable transfer pipettes Forceps Gloves powder free nitrile Laboratory grade wipes Lint free wipes Pipette tips 10 to 100 uL MLS Pipettes P10 to P1000 Plastic bins 3 medium to larget TE Buffer 1X Molecular Biology Grade Vortexer Water DNase free sterile filtered Optional Filtered 10096 compressed nitrogen gas or residue free compressed air canistert t For washing the tip blocks and plate holders t For drying the plate holder tip blocks and plate guides Applied Biosystems OpenArray9 Real Time PCR System Digital PCR User Guide Chapter 1 Overview Workflow Workfl
13. Applied Biosystems OpenArray9 Real Time PCR System Digital PCR User Guide CHAPTER 4 Troubleshooting The TaqMan Assays are optimized for use with OpenArray digital PCR reagents OpenArray instruments and thermal cycling conditions If you experience problems with assay performance be sure that you have followed our protocols then check the troubleshooting table below Observation Possible cause Recommended solution High fluorescence signal in the NTCs Non specific probe cleavage Perform proper bioinformatics on the sequence evaluate the assay design and consider redesigning the assay The NTC is contaminated Examine other assays for high fluorescence signal in the NTCs Consider replacing the water used for the NTCs the water may be a possible source of contamination The probe is degraded Store the assays correctly Unfilled through hole 1 In the OpenArray Digital PCR Software select the Heatmap tab 2 From the Data to Display drop down list select Amplification then use the heatmap to determine the location of the unfilled through holels 3 Visually inspect the array to confirm the existence of the unfilled through holes Amplification curve shows abnormal plot The baseline was set improperly some samples have Cy values lower than the baseline stop value Refer to your real time PCR system user guide for procedures on setting the baseline Switc
14. C7 data calculation The DNA polymerase cleaves only probes that are hybridized to the target Figure 4 Cleavage separates the reporter dye from the quencher dye the separation of the reporter dye from the quencher dye results in increased fluorescence by the reporter The increase in fluorescence occurs only if the target sequence is complementary to the probe and is amplified during PCR Because of these requirements nonspecific amplification is not detected Figure 4 Cleavage Forward CR x TaqMan Primer MGB probe 5 3 3 n 5 5 3 P 5 Reverse Primer Polymerization of the strand continues but because the 3 end of the probe is blocked no extension of the probe occurs during PCR Figure 5 Figure 5 Completion of polymerization MGB probe Forward d p Primer 3 qM d 3 V 5 5 3 5 Reverse Primer Threshold cycle C data calculation After thermal cycling the OpenArray Software uses a Cy confidence algorithm to calculate a threshold cycle Cy value for each through hole reaction on the OpenArray Digital PCR Plate Following the analysis the analyzed experiment data are exported as a comma separated value csv file using the OpenArray software Applied Biosystems OpenArray9 Real Time PCR System Digital PCR User Guide 59 Appendix C Background Information Quantitative analysis and copy number calculation Quantitative analysis and copy number calculation Presen
15. Confidence The software calculates calls using a combination of the Ct Range and Ct Confidence Range methods The software assigns positive calls to all through hole reactions that yield threshold cycles Cy that are both above the defined limit and within the defined range of valid Cys For each through hole position the software then assigns either a positive 1 call to the position if the Cr falls within the acceptable range or a negative 0 call if it falls outside During the final phase of the analysis the OpenArray Digital PCR Software generates copy number values and confidence interval data for each sample on the OpenArray Digital PCR Plate For each sample the software begins by calculating a copy number value for each subarray by counting the number of positive calls 1 Using the call data the OpenArray Digital PCR Software then calculates copy number values for all samples present on the plate and generates 9576 confidence intervals according to a Poisson maximum likelihood algorithm Fazekas de St Groth S 1982 Applied Biosystems OpenArray Real Time PCR System Digital PCR User Guide This appendix covers Symbols on instruments ssassn aseeseen rere General instrument safety a na aanas urrera Physical hazard safety 0 cece cece eee eee Electrical safety 2 02 20s seca agg bole ooo seas lee meee eens Workstation safety sese eee eens Safety and electromagnetic compatibility st
16. Details table Displays the subarray data for all OpenArray Digital PCR Plates loaded by the software Each row of the table displays the data for a specific subarray on the associated plate e OpenArray SN The serial number of the associated OpenArray Digital PCR Plate e ID The coordinates row and column of the subarray e Sample The sample loaded to the subarray Assay The TaqMan assay loaded to the subarray Dilution The dilution point of the sample loaded to the subarray 8 Q Reload Reverts the sample assay and dilution settings of the OpenArray Digital PCR Plates to the original settings 9 Accept Changes Saves the changes to the sample assay and dilution settings then closes the dialog box 10 3 Reject Changes Closes the dialog box without saving the sample assay and dilution settings 30 Applied Biosystems OpenArray9 Real Time PCR System Digital PCR User Guide Chapter3 Analyze the Results 3 Edit the experiment information Change the 1 Inthe OpenArray Digital PCR Software window select Display Sample Assay sample assay and Dilution Editor dilution settings 2 In the OpenArray Plate Sample Assay and Dilution Editor dialog box edit the sample assay and dilution data for the subarrays from the plate a Select the subarrays in each OpenArray Digital PCR Plate that you want to modify Note To select multiple subarrays click and drag across the subarrays
17. In the Heat Map tab of the OpenArray Digital PCR Software window place the mouse cursor over a unit on the z axis scale to the left of the heat map plot then click and drag to the maximum or minimum extreme of the plot Repeat to move the remaining units Change the display settings 1 In the OpenArray Digital PCR Software window select Advanced Display Settings or right click the plot then select Edit Display Options from the contextual menu 2 In the OpenArray Digital PCR Software Display Settings dialog box edit the analysis settings as needed Ci OpenArray Digital PCR D Heat Map Settings e Display Z Axis in Color Select to display the intensity of the heat map in color Deselect to beni duis display the heat map in grey scale Display Subarray Labels A j Display Through Hole Grid e Display Subarray Grid Select to use grid lines to Synchronize XY Axes define the subarrays in the heat maps of the OpenArray Digital PCR Plates Display Subarray Labels Select to display the coordinate positions of the subarrays in the plates Display Through Hole Grid Select to use grid lines to define the individual through hole positions in the heat maps of the plates e Synchronize XY Axes Select to lock the scaling of the X and Y axes so that they zoom synchronously If deselected the axes scale independently 3 When you are finished editing the display settings click Accept to apply
18. apply the dilution to the selected subarray s The value represents the dilution point of the corresponding sample where larger values indicate increasingly smaller dilutions of the sample For example the values 1 0 0 5 0 25 0 125 and 0 0625 can be used to represent a five point dilution series of 1 1 2 1 4 1 8 and 1 16 4 E Apply to Selected Applies the current data in the Sample Assay and Dilution fields to the selected subarray s 5 ty Clear Selected Removes the sample assay and dilution data from the selected subarray s 6 Plate grid Displays the grid of subarrays and assigned content in the first three OpenArray plates loaded by the software The software displays the row and column coordinates within each subarray You can use the plate layout as a selection tool to assign well contents and to view well assignments Note The editor displays only the first three OpenArray Digital PCR Plates that are loaded for analysis selected Subarray Sample Assigned OpenArray SN ID Assay Es 43 Al SRY Es 43 A2 SRY ESV43 A3 SRY ESV43 A4 SRY ESV43 AS SRY ESV43 SRY ESV43 RNaseP ESV43 RNaseP ESV43 RNaseP ESV43 RNaseP ES 43 RNaseP ESv43 RNaseP Es 43 SRY ESv43 SRY ESv43 SRY ESv43 SRY Dilution 0 250000 0 250000 0 500000 0 500000 1 000000 1 000000 0 250000 0 250000 0 500000 0 500000 1 000000 1 000000 0 250000 0 250000 0 500000 0 500000 v 7 Subarray Through Hole
19. for that same sample A dye that produces fluorescence signal independent of PCR amplification and that is added to each reaction at a constant concentration Because the passive reference signal should be consistent across all wells it is used to normalize the reporter dye signal to account for non PCR related fluorescence fluctuations caused by minor well to well differences in volume Normalization to the passive reference signal generally results in data with noticeably high precision among technical replicates An illustration of the grid of wells and assigned content in the reaction plate The number of rows and columns in the grid depends on the sample block that you use In the software you can use the plate layout as a selection tool to assign well contents to view well assignments and to view results The plate layout can be printed included in a report exported and saved as a slide for a presentation A file csv that contains setup information such as the well number sample name sample color target name dyes and other reaction plate contents PCR reaction component that contains the primers designed to amplify the target and a TaqMan probe designed to detect amplification of the target The amount of target in the samples Absolute quantity can refer to copy number mass molarity or viral load A molecule attached to the 3 end of TaqMan probes to prevent the reporter from emitting fluorescence signal while t
20. for the confidence experiment using the new analysis settings interval calculated for the threshold cycles C of all through hole reactions The software assigns positive calls to all through hole reactions that yield threshold cycles C1 that are above the limit defined in the Minimum Ct Confidence field 5 Reject Closes the dialog box without applying the changes to the analysis settings e Ct and Confidence The software calculates calls using a combination of the Ct Range and Ct Confidence Range methods The software assigns positive calls to all through hole reactions that yield threshold cycles Cy that are both above the defined limit and within the defined range of valid Crs 38 Applied Biosystems OpenArray Real Time PCR System Digital PCR User Guide Chapter 3 Analyze the Results 3 Save and export the results Change the 1 In the OpenArray Digital PCR Software window select Advanced gt Analysis analysis settings Settings 2 In the OpenArray Digital PCR Software Analysis Settings dialog box edit the analysis settings as needed If you change the Cy calculation method you can use the Cy Confidence vs Cy Plot to understand how your choice of method affects the calls The OpenArray Digital PCR Software updates the plot in real time as you modify the analysis settings 3 When you are finished editing the display settings click Accept to apply the changes you have made Save and export the re
21. gt applied pine eteme by lfe technologies OpenArray Real Time PCR System Digital PCR Experiments User Guide For Research Use Only Not intended for any animal or human therapeutic or diagnostic use Information in this document is subject to change without notice APPLIED BIOSYSTEMS DISCLAIMS ALL WARRANTIES WITH RESPECT TO THIS DOCUMENT EXPRESSED OR IMPLIED INCLUDING BUT NOT LIMITED TO THOSE OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE TO THE FULLEST EXTENT ALLOWED BY LAW IN NO EVENT SHALL APPLIED BIOSYSTEMS BE LIABLE WHETHER IN CONTRACT TORT WARRANTY OR UNDER ANY STATUTE OR ON ANY OTHER BASIS FOR SPECIAL INCIDENTAL INDIRECT PUNITIVE MULTIPLE OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT INCLUDING BUT NOT LIMITED TO THE USE THEREOF WHETHER OR NOT FORESEEABLE AND WHETHER OR NOT APPLIED BIOSYSTEMS IS ADVISED OF THE POSSIBILITY OF SUCH DAMAGES NOTICE TO PURCHASER Label License No right to resell this product or any of its components is conveyed expressly by implication or by estoppel This product is for internal research purposes only and is not for use in commercial services of any kind including without limitation reporting the results of purchaser s activities for a fee or other form of consideration For information on obtaining additional rights please contact outlicensing dlifetech com or Out Licensing Life Technologies 5791 Van Allen Way Carlsbad California 2008
22. in data analysis When adding NTCs to the OpenArray 384 Well Sample Plate place one NTC in each section of the stock plate to ensure that the NTCs are plated in the correct location in the OpenArray Digital PCR Plate Note The NTC rate for assays of interest can be characterized on the first few OpenArray Digital PCR Plates of an experiment and excluded from the remaining plates The nature of digital PCR places constraints on the number of NTCs per run Technical We recommend the use of technical replicates to provide optimal confidence intervals replicates for the digital PCR analysis By default an OpenArray Digital PCR Plate provides 64 technical replicates per loaded sample assay combination resulting from the loading of a single subarray Increasing the number of replicate subarrays loaded with the same sample assay combination provides increasingly narrower confidence intervals When selecting the number of replicates to use per sample assay combination in a digital PCR experiment select the number of replicates at which the benefit of adding more falls below a target percentage Applied Biosystems OpenArray Real Time PCR System Digital PCR User Guide 51 Appendix B Example Layouts Example plate layout 1 Example plate layout 1 This section describes an example plate layout for a digital PCR experiment that evaluates a broad dilution range of two samples for a single nucleic acid target Description In this example
23. intensities Samples with lower starting quantities exponentially amplify lower yields compared to samples with higher starting quantities Use a high quality spectrophotometer or perform an RNase P quantitation assay to determine the concentration of each sample Normalize as needed Refer to the User Bulletin Human DNA Sample Quantification Protocol Using the RNase P Kit available from the Applied Biosystems website Pipetting errors Poorly calibrated pipettes incorrect pipette tips or inefficient technique result in varied volumes pipetted into the sample plate and in varied genomic DNA concentrations Ensure that all pipettes are calibrated on a routine basis and use the recommended pipette tips Consult the pipette manufacturer for proper testing and maintenance Check the ROX dye levels Variation in the ROX dye levels may indicate pipetting errors Expired reagents Replace with fresh reagents Evaporation has occurred prior to loading the plate in the case Check the ROX dye levels after imaging Variation in the ROX dye levels may indicate evaporation PCR inhibitors ranging from organics to non organics can cause samples to fail amplification Examine the purity of the DNA by checking the e A240 A2g0 ratio which should be between 1 7 and 1 9 A ratio 1 7 indicates protein contamination A240 A239 ratio which should be similar to the Aygo A gp ratio A ratio 1 7 i
24. of copies for each sample or replicate group For each data point the plot displays error bars that indicate the upper and lower limits of the associated confidence interval calculated by the software Note See Change the plot scale below to scale the plot 5 Annotation On checkbox Select to display sample and assay names within the Bar Plot of Copies Per Reaction 6 Legend Displayed checkbox Select to display the legend for the Bar Plot of Copies Per Reaction 7 Crosshair tool Select to change the mouse cursor to a crosshair which provides accurate selection of data Guidelines for reviewing the results 8 39 Magnifier tool Opens a menu of magnification tools for manipulating the plot Zooms the plot to the selected region Zooms the plot horizontally to the selected region without expanding the vertical axis Zooms the plot vertically to the selected region without expanding the horizontal axis Resets the view to display the entire plot xe Zooms out the plot incrementally with each click e Zooms in the plot incrementally with each click 9 W Grab tool Select to change the mouse cursor to a hand which allows manual repositioning of the plot Review the results data for the following Review the error bars for each sample Large error bars can indicate that too few technical replicates were loaded for the associated sample or that the dilution of the associated
25. on the plate b In the Sample Assay and or Dilution fields enter the values that you want to apply to the selected subarrays c Apply the values to the selected subarrays Either e Click 5 next a field to apply the associated value Click Apply to Selected to apply all sample assay and dilution values Note Click Clear Selected to remove the sample assay and dilution values from the selected subarrays Note Click Q Reload to revert the sample assay and dilution values of the loaded OpenArray Digital PCR Plates to the original settings 3 Repeat step 2 as needed to edit the sample assay and dilution data for the OpenArray Digital PCR Plates 4 When you are finished editing the sample assay and dilution data click Q9 Accept Changes to apply the changes that you have made Applied Biosystems OpenArray9 Real Time PCR System Digital PCR User Guide 31 Chapter3 Analyze the Results View the results View the results After you load a raw amplification curve data csv file and edit the experiment information review the results of the default analysis in the Results tab as explained below About the Results tab The Results tab allows you to view the results of the digital PCR experiments in tabular and graphical formats About the Results table Heat Map Results Zi Display Results by Replicate Group v Group Average Lower Upper Total Total Total Confidence Interval CopiesPer Conf
26. sample preparation and template quality must be assessed on an individual basis Make sure that the DNA you use for experiments e Is extracted from the raw material that you are testing with an optimized protocol salting out procedures and crude lysates are not recommended Does not contain PCR inhibitors e Has A260 230 and A260 280 ratios between 1 7 and 1 9 e Is intact as visualized by gel electrophoresis Has not been heated above 60 C temperatures above 60 C can cause degradation The quantity of sample added to a digital PCR reaction depends on the Amount of genomic or complementary DNA gDNA or cDNA present in each sample Number of copies of the target sequence present in the genome of your samples Quantitation methods Before performing digital PCR experiments consider quantifying the amount of gDNA or cDNA in each sample We recommend the following methods of quantitation e Quant iT assay nucleic acid quantitation using the Qubit Quantitation Platform or e Real time PCR using your own DNA samples or TaqMan DNA Template Reagents PN 401970 to create a standard curve Refer to Creating Standard Curves with Genomic DNA or Plasmid DNA Templates for Use in Quantitative PCR PN 4371090 for more information You can download the document from docs appliedbiosystems com search taf Applied Biosystems OpenArray Real Time PCR System Digital PCR User Guide Chapter 2 Prepare and Perform the Digital PCR Expe
27. the changes you have made Applied Biosystems OpenArray9 Real Time PCR System Digital PCR User Guide 35 3 Chapter 3 Analyze the Results Review and omit outliers Review and omit outliers About outliers About the auto omission algorithm 36 After reviewing the results of the analysis you can use the OpenArray Plate Omission Editor to manually omit well data from the analysis if necessary Outliers occur when a factor other than initial sample quantity affects the PCR amplification and the measured Cy value Outliers can result from either random experimental error or a number of laboratory errors such as contamination plate seal leaks pipetting inaccuracies or instrument issues Note We recommend manually removing outliers only when the rationale to remove a technical replicate well is objective and obvious The OpenArray Digital PCR Software uses an algorithm to identify and remove from the analysis through holes that were not adequately filled with reaction mix The software uses multiple spectral channels from the preamplification fluorescence signal to identify inadequate fills on the OpenArray plate where abnormally low signal can indicate improper filling of a through hole Prior to thermal cycling the algorithm establishes a threshold for an array by analyzing multiple spectral channels of the preamplification data from all through holes on the array The software then compares the preamplification data of individu
28. the World Health Organization WHO Laboratory Biosafety Manual third edition http www who int csr resources publications biosafety WHO CDS CSR LYO 2004 1l en Chemical alerts General alerts for Avoid contact with skin eyes and or clothing Read the MSDS and follow the all chemicals handling instructions Wear appropriate protective eyewear clothing and gloves Specific CAUTION CHEMICAL HAZARD TaqMan OpenArray Digital PCR chemical alerts Master Mix may cause eye and skin irritation 70 Applied Biosystems OpenArray Real Time PCR System Digital PCR User Guide Bibliography Afonina I Zivarts M Kutyavin 1 et al 1997 Efficient priming of PCR with short oligonucleotides conjugated to a minor groove binder Nucleic Acids Res 25 2657 2660 Fazekas de St Groth S 1982 The Evaluation of Limiting Dilution Assays J Immunol Meth 49 R11 22 Forster V T 1948 Zwischenmolekulare Energiewanderung und Fluoreszenz Annals of Physics Leipzig 2 55 75 Kutyavin I V Lukhtanov E A Gamper H B and Meyer R B 1997 Oligonucleotides with conjugated dihydropyrroloindole tripeptides base composition and backbone effects on hybridization Nucleic Acids Res 25 3718 3723 Lakowicz J R 1983 Energy Transfer In Principles of Fluorescence Spectroscopy New York Plenum Press 303 339 Longo M C Berninger M S and Hartley J L 1990 Use of uracil DNA glycosylase to control carryover contaminati
29. then perform the run BR WO N 5 Optional Enter sample assay and dilution information using the OpenArray software l Applied Biosystems OpenArray Real Time PCR System Digital PCR User Guide 15 1 Chapter 1 Overview Workflow Review and export the run data 1 View the results 2 Optional Modify project files ncx 3 Export the raw amplication data to a comma separated value file csv l Analyze the data using the OpenArray Digital PCR Software Open the experiment Edit the experiment information View the results View the data Optional Optimize the analysis settings noo PF C N Save and export the results 16 Applied Biosystems OpenArray9 Real Time PCR System Digital PCR User Guide CHAPTER 2 Prepare and Perform the Digital PCR Experiments In this chapter Prepare the DNA samples ssssssesssseee e 18 Prepare the digital PCR experiment ssssseeeeeeeeee 20 Perfor the f ns 20osLcoesorepeIELOMP RIAM pL EHE DUE gate d bd 23 Applied Biosystems OpenArray9 Real Time PCR System Digital PCR User Guide 17 2 Chapter 2 Prepare and Perform the Digital PCR Experiments Prepare the DNA samples Prepare the DNA samples Quality of DNA Quantity of DNA 18 We recommend the following best practices for the preparation of DNA template for use in digital PCR experiments Because digital PCR experiment strategy and methodology can vary significantly
30. weight of the computer and or the monitor moving them may require two or more people Things to consider before lifting the computer and or the monitor Make sure that you have a secure comfortable grip on the computer or the monitor when lifting Make sure that the path from where the object is to where it is being moved is clear of obstructions Donotlift an object and twist your torso at the same time Keep your spine in a good neutral position while lifting with your legs Participants should coordinate lift and move intentions with each other before lifting and carrying Instead of lifting the object from the packing box carefully tilt the box on its side and hold it stationary while someone slides the contents out of the box Ensure that anyone who operates the instrument has Received instructions in both general safety practices for laboratories and specific safety practices for the instrument Read and understood all applicable Safety Data Sheets SDSs See About SDSs on page 68 Applied Biosystems OpenArray9 Real Time PCR System Digital PCR User Guide 63 Appendix D Safety Physical hazard safety Cleaning or CAUTION Before using a cleaning or decontamination method other than decontaminating those recommended by the manufacturer verify with the manufacturer that the the instrument proposed method will not damage the equipment Physical hazard safety Ultraviolet light AN WARNING ULTRAVI
31. OLET LIGHT HAZARD Looking directly at a UV light source can cause serious eye damage Never look directly at a UV light source and always prevent others from UV exposure Follow the manufacturer s recommendations for appropriate protective eyewear and clothing Moving parts WARNING PHYSICAL INJURY HAZARD Moving parts can crush and cut Keep hands clear of moving parts while operating the instrument Disconnect power before servicing the instrument Electrical safety WARNING ELECTRICAL SHOCK HAZARD Severe electrical shock can result from operating the OpenArray Digital PCR Software without its instrument panels in place Do not remove instrument panels High voltage contacts are exposed when instrument panels are removed from the instrument Fuses WARNING FIRE HAZARD Improper fuses or high voltage supply can damage the instrument wiring system and cause a fire Before turning on the instrument verify that the fuses are properly installed and that the instrument voltage matches the power supply in your laboratory WARNING FIRE HAZARD For continued protection against the risk of fire replace fuses only with fuses of the type and rating specified for the instrument 64 Applied Biosystems OpenArray9 Real Time PCR System Digital PCR User Guide Appendix D Safety Workstation safety Power WARNING ELECTRICAL HAZARD Grounding circuit continuity is required for the safe operation of equipment Never operate equipment wi
32. PCR Plate data chosen in the Data to Display drop down list The software displays heat maps for the first three OpenArray9 Digital PCR Plates that are loaded for analysis For each plate the software displays the serial number of the plate to the left of the heat map and the legend for the heat map to the right Note See Change the appearance of the heat map on page 35 to use the OpenArray Real Time PCR System Display Settings dialog box to change the appearance of the heat maps 34 Applied Biosystems OpenArray Real Time PCR System Digital PCR User Guide Guidelines for reviewing the Heat Map tab Change the appearance of the heat map Chapter3 Analyze the Results 3 View the data e Use the Omitted heat map plot to locate the subarrays that were automatically omitted by the OpenArray software then use the Amplification heat map plot to review the amplification data for the omitted subarrays Review the heat map data for the following Review all replicate groups to confirm that replicate subarrays exhibit roughly equivalent amplification Confirm that the subarrays containing negative controls do not amplify Confirm that all subarrays contain at least some negative calls Subarrays that do not contain any negative calls indicate that the sample concentration is too great The OpenArray Digital PCR Software provides two ways to adjust the appearance of the heat maps Change the color scale of the z axis
33. PCR System Digital PCR User Guide Chapter 1 Overview 1 Introduction OpenArray Digital OpenArray Digital PCR Software performs a copy number and Poisson statistical PCR Software analysis of the digital PCR experiments performed using TaqMan assays on an OpenArray Real Time PCR System The software can be used to detect and measure copy number of specific sequences in a variety of samples Applications of digital PCR include quantitation of low level pathogens rare genetic sequences gene expression in single cells and low fold copy number discrimination of genes targets Features The OpenArray Digital PCR Software can e Be used to plan digital PCR experiments The Poisson calculator can be used to calculate the expected copy number per reaction including confidence intervals from user specified number of replicates and negative calls e Open and analyze OpenArray Real Time PCR System experiment data Calculate positive negative amplification calls using four available methods default algorithm Cy Range Cr Confidence Cr Range and Confidence Modify sample and assay data applied to digital PCR experiments run e Omit outlier through hole reactions from the analysis Report confidence in copy number calls Display calculated sample copy number data in both tabular and graphic formats View detailed data analysis information Change analysis parameters and reanalyze the data e Simultaneously analyze an
34. R Master Mix 10 times Applied Biosystems OpenArray Real Time PCR System Digital PCR User Guide Load the OpenArray Digital PCR Plates Chapter 2 Prepare and Perform the Digital PCR Experiments Prepare the digital PCR experiment 5 Transfer the master mix TaqMan assay and DNA samples to the OpenArray 384 Well Sample Plate IMPORTANT The component amounts vary depending your experiment layout Note When preparing master mix for multiple OpenArray Digital PCR Plates adjust the volumes accordingly Volume pL Material Stock Final Subarray Platet TaqMan OpenArray 2 5 180 2X 1X Digital PCR Master Mix 2X TaqMan Assay 20X 0 25 18 20X 1X primer probe mix Diluted DNA 2 144 0 15 ng uL 0 06 ng uL Water 0 25 18 Total volume 5 5 360 t Per OpenArray Digital PCR Plate volumes include 3096 excess for volume loss from pipetting 6 Mix well by gently pipetting up and down 7 Cover the OpenArray Sample Plate with sealing tape 8 Centrifuge the OpenArray Sample Plate for 1 minute at 1000 rpm to eliminate bubbles from the wells IMPORTANT For optimal results we recommend that you load OpenArray Sample Plates within an hour after you prepare them Use the OpenArray AutoLoader to transfer the reactions from the OpenArray 384 Well Sample Plate to an OpenArray Digital PCR Plate Refer to the TagMan OpenArray Real Time PCR Plates Pro
35. R System 1 Close the OpenArray instrument lid and door 2 In the Input Plate Serial Numbers dialog box click Cycle to begin the run During thermal cycling the OpenArray instrument records the amount of fluorescence from each through hole of the OpenArray Digital PCR Plates at each cycle of the PCR The OpenArray software automatically saves the run data to the associated plate data file tpd IMPORTANT Do not open the instrument door during the run The run is complete when the blue LED light on the instrument door is off and the OpenArray software displays data and a green circle in the status bar 3 When the run is complete save the project file ncx a Select File Save or File Save As to open a save dialog box b Browse to a save location enter a file name then click Save 4 Open the instrument door then remove the OpenArray Digital PCR Plates When the real time imaging run is complete the OpenArray software automatically analyzes the data for each OpenArray Digital PCR Plate If desired you can review the results of the automatic analysis in the Assays pane the Sample Data pane and the Curve pane If the analysis settings are not acceptable for your experiment you can modify the data by normalizing the data setting outliers or by adjusting the CT settings Applied Biosystems OpenArray Real Time PCR System Digital PCR User Guide Chapter 2 Prepare and Perform the Digital PCR Experimen
36. TANT Indicates information that is necessary for proper instrument operation accurate chemistry kit use or safe use of a chemical CAUTION Indicates a potentially hazardous situation that if not avoided AN may result in minor or moderate injury It may also be used to alert against unsafe practices WARNING Indicates a potentially hazardous situation that if not avoided could result in death or serious injury DANGER Indicates an imminently hazardous situation that if not avoided will result in death or serious injury This signal word is to be limited to the most extreme situations Except for IMPORTANTS each safety alert word in an Applied Biosystems document appears with an open triangle figure that contains a hazard symbol These hazard symbols are identical to the hazard symbols that are affixed to Applied Biosystems instruments see Safety symbols on page 62 The safety data sheets SDSs for any chemicals supplied by Applied Biosystems or Ambion are available to you free 24 hours a day For instructions on obtaining SDSs see SDSs on page 68 IMPORTANT For the SDSs of chemicals not distributed by Applied Biosystems or Ambion contact the chemical manufacturer Applied Biosystems OpenArray9 Real Time PCR System Digital PCR User Guide In this chapter Applied Biosystems OpenArray9 Real Time PCR System Digital PCR User Guide Introduction eero rani 00 cece eee Install the software
37. User s Guide EMC This OpenArray AccuFill System OpenArray Case Sealing Station and OpenArray Instrument meets European requirements for emission and immunity EMC Directive 2004 108 EC This instrument has been tested to and complies with standard EN 61326 Group 1 Class A Electrical Equipment for Measurement Control and Laboratory Use EMC Requirements Australia and New This OpenArray AccuFill System OpenArray Case Sealing Station and Zealand EMC OpenArray Instrument have been tested to and comply with standard AS NZS 2064 standards Limits and Methods Measurement of Electromagnetic Disturbance Characteristics of Industrial Scientific and Medical ISM Radio frequency Equipment 66 Applied Biosystems OpenArray Real Time PCR System Digital PCR User Guide Appendix D Safety General chemical safety General chemical safety Chemical hazard warning WARNING CHEMICAL HAZARD Before handling any chemicals refer to the Safety Data Sheet SDS provided by the manufacturer and observe all relevant precautions WARNING CHEMICAL HAZARD All chemicals in the instrument are potentially hazardous Always determine what chemicals have been used in the instrument before changing reagents or instrument components Wear appropriate eyewear protective clothing and gloves when working on the instrument d WARNING CHEMICAL HAZARD Four liter reagent and waste bottles can crack and leak
38. a for the loaded results file see page 32 for more information Applied Biosystems OpenArray9 Real Time PCR System Digital PCR User Guide ID The coordinates row and column of the through holes in the subarray Ct The threshold cycle C of the associated through holes Ct Confidence The confidence of the Cy calculated for the associated through holes The positive 1 or negative 0 call applied to the through holes of the subarray 29 Chapter 3 Analyze the Results Edit the experiment information Edit the experiment information After you load a raw amplification curve data csv file you can use the OpenArray Plate Sample Assay and Dilution Editor to add or modify the sample assay and dilution assignments applied to the OpenArray Digital PCR Plate data About the editor The OpenArray Plate Sample Assay and Dilution Editor allows you to change the sample assay and dilution assignments applied to the OpenArray digital PCR experiments The elements in the following figure are described below Editor Actions Sample Dilution t Apply to Selected Clear Selected 1 Sample field Enter a sample name then click to apply the name to the selected subarray s 2 Assay field Enter an assay name then click to apply the assay to the selected subarray s 3 Dilution field Enter a dilution point integer or floating point number then click 5 to
39. al through holes to the calculated threshold omitting those with signal below the limit To ensure that a failed through hole is not a single measurement discrepancy the software only omits a through hole if it produces multiple fluorescent measurements that fail the threshold criteria Applied Biosystems OpenArray9 Real Time PCR System Digital PCR User Guide Chapter 3 Analyze the Results Review and omit outliers The OpenArray Plate Omission Editor allows you to omit data from the analysis The elements in the following figure are described below About the editor OpenArray Plate Omission Editor Open rray Plate Serial Number 1 OpenArray Plate Serial Number drop down list Selects the plate data displayed by the OpenArray Plate Omission Editor The list contains the barcodes of all plates included in the experiment Note A check mark appears next to the displayed plate 2 Plate grid Displays the grid of subarrays and through Omitted Through Hole Open rray Plate eie46 Subarray A12 3 Reload Reverts the OpenArray Digital PCR Plates to their original settings 4 Accept Changes Saves the changes to the omission settings then closes the dialog box 5 Reject Changes Closes the dialog box without saving the omission settings hole positions on the OpenArray Digital PCR Plate In the OpenArray Digital PCR Software window select Display OpenArray Plate Om
40. also cycling stage In a PCR reaction mix two target specific primers or two primers and a probe used to amplify a target Identifier assigned by Applied Biosystems to TaqMan assays Tab delimited data file on a CD shipped with each assay order The AIF contains technical details about all assays in the shipment It includes information about assay concentrations reporters and quenchers used part and lot numbers and assay vial and plate ID numbers The file name includes the number from the bar code on the plate In the amplification plot a cycle to cycle range that defines background fluorescence This range can be set manually on an assay by assay basis or automatically to set each individual well See reagents See threshold cycle CT Algorithm used to determine the threshold cycle Applied Biosystems OpenArray9 Real Time PCR System Digital PCR User Guide 73 Glossary cycling stage data collection diluent export forward primer holding stage import manual baseline manual threshold multicomponent plot negative control NC no template control NTC nonfluorescent quencher minor groove binder NFQ MGB 74 In the thermal profile a stage that is repeated A cycling stage is also called an amplification stage See also amplification stage During the instrument run a process in which an instrument detects fluorescence data from each well of the reaction plate The instrument t
41. andards General chemical safety 6 2 00 066 c cece eee eens Chemical waste safety 0 000 c cece eee es Biological hazard safety Chemical al rts esce uber ER Applied Biosystems OpenArray Real Time PCR System Digital PCR User Guide APPENDIX D Safety 61 Appendix D Safety Symbols on instruments Symbols on instruments Electrical symbols on instruments Safety symbols Environmental symbols on instruments 62 The following table describes the electrical symbols that may be displayed on Applied Biosystems instruments Symbol Description Symbol Description Indicates the On position of the main power switch Indicates the Off position of the main power switch Indicates a protective grounding terminal that must be connected to earth ground before any other electrical connections are made to the instrument O Indicates a terminal that may be connected to the signal ground reference of another instrument This is not a protected ground terminal Indicates a terminal that can eu f receive or supply alternating current or voltage emmp indicates that the device eco receives or supplies direct current or voltage The following table describes the safety symbols that may be displayed on Applied Biosystems devices Each symbol may appear by itself or with text that explains the relevant hazard These safety symbols may als
42. ation on how to perform digital PCR experiments on the OpenArray Real Time PCR System and how to analyze the resulting data using the OpenArray Digital PCR Software Audience This user guide is written for principal investigators and laboratory staff who perform digital PCR nucleic acid quantitation using the OpenArray9 Real Time PCR System and the OpenArray Digital PCR Software Assumptions This guide assumes that you have e Familiarity with Microsoft Windows operating system Knowledge of techniques for handling and preparing DNA samples for PCR e A general understanding of data storage file transfers and copying and pasting e Access to the example experiment provided with the OpenArray Real Time PCR System Applied Biosystems OpenArray Real Time PCR System Digital PCR User Guide 5 About This Guide Safety information Safety information Safety alert words Safety data sheets Note For general safety information see this section and Appendix D Safety on page 61 When a hazard symbol and hazard type appear by a chemical name or instrument hazard see the Safety Appendix for the complete alert on the chemical or instrument Four safety alert words appear in Applied Biosystems user documentation at points in the document where you need to be aware of relevant hazards Each alert word IMPORTANT CAUTION WARNING DANGER implies a particular level of Observation or action as defined below IMPOR
43. ature Tn without increasing probe length Afonina et al 1997 Kutyavin et al 1997 they also allow for the design of shorter probes Anonfluorescent quencher NFQ at the 3 end of the probe Because the quencher does not fluoresce OpenArray Systems can measure reporter dye contributions more accurately For more information see PCR and the 5 nuclease assay on page 58 e Technical replicates Through hole reactions of each subarray that contain identical sample assay reaction mix combinations and volumes Each subarray of the OpenArray Digital PCR Plate contains a minimum of 64 technical replicates resulting from a single well of the 384 well sample plate See Technical replicates on page 51 for a complete discussion of replicates e Optional No template controls NTCs Samples that contain water or buffer instead of template also known as negative controls NICs should not amplify See No template controls on page 51 for a complete discussion of no template controls About digital PCR experiment setup In a digital PCR experiment performed on an OpenArray System dilutions of each gDNA or cDNA sample are loaded into the wells of an OpenArray 384 Well Sample Plate that contain TaqMan OpenArray Digital PCR Master Mix and TaqMan assay The samples are diluted down to a limiting quantity such that most individual PCR reactions contain either zero or one target molecules Applied Biosystems OpenArray9 Real Time
44. ce absence call generation Poisson analysis and copy number calculation 60 The OpenArray9 Digital PCR Software performs a quantitative analysis of the digital PCR experiment data obtained by the OpenArray System during the PCR When the exported data is loaded the OpenArray Digital PCR Software automatically performs the phased analysis of the experiment data described below During the second phase of the analysis the OpenArray Digital PCR Software uses the calculated threshold cycle Cy data to generate a presence absence call for each through hole position The software determines the calls by employing one of four methods to establish limits that define the valid range of Crs for making positive calls Default The software calculates calls using a proprietary Life Technologies Corporation algorithm Ct Range The software calculates calls using a discrete range of PCR cycles The software assigns positive calls to all through hole reactions that yield threshold cycles Cy that are within the range defined in the Minimum Ct and Maximum Ct fields e Ct Confidence Range The software calculates calls using a user defined minimum limit for the confidence interval calculated for the threshold cycles Cy of all through hole reactions The software assigns positive calls to all through hole reactions that yield threshold cycles Cy that are above the limit defined in the Minimum Ct Confidence field Ctand
45. d Information In this appendix PCR and the 5 nuclease assay 6 eee eene 58 Quantitative analysis and copy number calculation 000008 60 Applied Biosystems OpenArray Real Time PCR System Digital PCR User Guide 57 Appendix C Background Information PCR and the 5 nuclease assay PCR and the 5 nuclease assay The 5 nuclease assay process Figures 2 through 5 takes place during PCR amplification This process occurs in every cycle and does not interfere with the exponential accumulation of product Figure 1 Legend for Figures 2 through 5 NFO Nonfluorescent quencher 468 Minor groove binder Reporter Hot start DNA polymerase During PCR the TaqMan MGB probe anneals specifically to a complementary sequence between the forward and reverse primer sites Figure 2 When the probe is intact Figures 2 and 3 the proximity of the reporter dye to the quencher dye results in suppression of the reporter fluorescence primarily by F rster type energy transfer Forster 1948 Lakowicz 1983 Figure 2 Polymerization Forward Primer 5 g E M 5 5 IZ Au F g P 5 Reverse Primer Figure 3 Strand displacement Forward TaqMan Primer MGB probe 5 y 3 A le SP 5 5 3 PO 5 Reverse Primer 58 Applied Biosystems OpenArray9 Real Time PCR System Digital PCR User Guide Appendix C Background Information Threshold cycle
46. d by Applied Biosystems contact the chemical manufacturer 68 Applied Biosystems OpenArray9 Real Time PCR System Digital PCR User Guide Appendix D Safety Chemical waste safety Chemical waste safety Chemical waste hazards CAUTION HAZARDOUS WASTE Refer to Safety Data Sheets and local regulations for handling and disposal WARNING CHEMICAL WASTE HAZARD Wastes produced by Applied Biosystems instruments are potentially hazardous and can cause injury illness or death WARNING CHEMICAL STORAGE HAZARD Never collect or store waste in a glass container because of the risk of breaking or shattering Reagent and waste bottles can crack and leak Each waste bottle should be secured in a low density polyethylene safety container with the cover fastened and the handles locked in the upright position Wear appropriate eyewear clothing and gloves when handling reagent and waste bottles Chemical waste To minimize the hazards of chemical waste safety guidelines Read and understand the Safety Data Sheets SDSs provided by the manufacturers of the chemicals in the waste container before you store handle or dispose of chemical waste Provide primary and secondary waste containers A primary waste container holds the immediate waste A secondary container contains spills or leaks from the primary container Both containers must be compatible with the waste material and meet federal state and local requirements for c
47. d view data from multiple OpenArray Plates Export data and graphs e Save or print analyzed data for further analysis or for sharing with colleagues that use the OpenArray Digital PCR Software Compatible instruments The OpenArray Digital PCR Software can be used to analyze the results of digital PCR experiments run on the OpenArray9 Real Time PCR System that have been exported as raw amplification curve data csv files About the analysis The OpenArray Digital PCR Software generates copy number data from fluorescence data collected from TaqMan reactions that have been loaded onto a OpenArray Digital PCR Plate and run on an OpenArray Real Time PCR System Following thermal cycling the raw amplification curve data from the digital PCR experiment are exported from the OpenArray software The exported file is then loaded by the OpenArray Digital PCR Software for analysis The OpenArray Digital PCR Software employs one of four methods to generate calls for all through hole reactions where reactions that exhibit amplification are assigned positive calls and those without amplification are assigned negative calls Using the call data the OpenArray Digital PCR Software calculates copy number values for all samples present on the plate and generates 9576 confidence intervals according to a Poisson maximum likelihood algorithm Fazekas de St Groth S 1982 Applied Biosystems OpenArray Real Time PCR System Digital PCR Use
48. e OpenArray Plates Chapter 1 Overview 1 Introduction The OpenArray System requires two plate types e OpenArray 384 Well Sample Plate sample plate e OpenArray Digital PCR Plate experiment plate OpenArray 384 Well Sample Plate The OpenArray 384 Well Sample Plate is a 384 well reaction plate You combine the TaqMan OpenArray Digital PCR Master Mix TaqMan assay and your DNA sample in the sample plate then use the OpenArray AutoLoader to transfer the mixture from the sample plate to an experiment plate s IMPORTANT The well dimensions of the OpenArray 384 Well Sample Plates are specifically suited for use with the OpenArray AutoLoader We do not recommend the use of other microtiter plates with the AutoLoader OpenArray Digital PCR Plate The OpenArray Digital PCR Plate is a 63 mm x 19 mm mid density reaction plate Each plate contains 3072 reaction through holes each of which can accommodate a 33 nL reaction volume As shown in the figure below the OpenArray Digital PCR Plate is divided into 48 subarrays where each subarray consists of 64 through holes Hydrophilic and hydrophobic coatings allow reagents to be held within the through holes Each subarray has these 64 through holes For example this 00000000 is A1g7 123456768 Applied Biosystems OpenArray9 Real Time PCR System Digital PCR User Guide 9 1 Chapter 1 Overview Introduction Digital PCR
49. e run Load the Note Refer to the OpenArray Real Time PCR System User Guide PN 4458837 for more Open Array Real information on loading the OpenArray Real Time PCR System Time PCR System 1 In the OpenArray Real Time PCR System Software open a project file ncx You can open e Anew project file Use the project file automatically opened at startup or select File New e An existing project file containing data from previous runs Select File Open then browse to and open a project file 2 Click Cycle 3 In the Position 1 field in the Input Plate Serial Numbers dialog box enter or scan the serial number for the first OpenArray Digital PCR Plate located on the package Alternately click Locate File then browse to and open the plate setup file tpf that corresponds to the OpenArray Digital PCR Plate IMPORTANT If you enter the serial number by typing or scanning the plate setup file tpf must be located at drive V Program Files BioTrove V PLATEFILES 4 Open the OpenArray instrument door and lid then place the OpenArray Digital PCR Plate into Position 1 Be sure that The plate position in the instrument matches the plate position in the OpenArray software The barcode is facing up and to the right and the plate is flush with the right and back edges IMPORTANT If the plates are not positioned correctly your data results will be adversely affected 5 Repeat this procedure
50. e sample plate is divided into eight areas where each area is 12 x 4 wells 48 wells During each load the OpenArray AccuFill System or the OpenArray AutoLoader transfers sample from one area of a sample plate to the corresponding position of the OpenArray Digital PCR Plate Plate Area 4 detail Columns 13 24 2 4 a B 10 42 14 18 18 20 22 24 EBBEBHEBEEBE Plate area 1 Plate area 2 Rows E H S a apoooood Em pEBBEBEBBEBH Plate area 3 Plate area 4 Columns 1 12 Columns 13 24 Rows A D Rows E H Rows I L Plate area 5 Plate area 6 on EU XU m mim Rows M P Plate area 7 Plate area 8 IMPORTANT The arrangement of samples and assays on the sample plates depends on the purpose of your digital PCR experiment The OpenArray Digital PCR Plate is a 63 mm x 19 mm mid density reaction plate Each plate contains 3072 reaction through holes each of which can accommodate a 33 nL reaction volume As shown in the following figure the OpenArray Digital PCR Plate is divided into 48 subarrays where each subarray consists of 64 through holes Hydrophilic and hydrophobic coatings allow reagents to be held within the through holes ENE REE Each subarray has these 64 through holes For example this is A1g7 12345678 Applied Biosystems OpenArray Real Time PCR System Digital PCR User Guide Appendix B Example Layouts Digital PCR experiment setup Digital PCR experiment s
51. ed Verify that the cDNA and TaqMan OpenArray Digital PCR Master Mix were added to the reaction plate If the master mix is missing the passive reference fails Incorrect dye components were selected Check the dye components settings and reanalyze the data The annealing temperature on the thermal cycler was too high for the primers and or probe Verify that the thermal cycler is set to the correct annealing and extension temperatures Ensure that the thermal cycler is calibrated and maintained regularly Inappropriate reaction conditions were used Troubleshoot the RT PCR optimization Degraded template Determine the quality of the template Rerun the assay with fresh template Use RNase free reagents Usean RNase inhibitor Inhibitors present in the reaction Verify the presence of an inhibitor 1 Create a serial dilution of your sample 2 Run the serial dilution with an expressing assay for example an endogenous control If an inhibitor is present low concentrations yield higher than expected Cy values High concentration means more inhibition because the sample is not diluted 3 Rerun the assay with purified template The baseline and or threshold was improperly set Refer to your real time PCR system user guide for procedures on setting the baseline and threshold Switch from automatic to manual baselining or from manual to automatic Lower the thre
52. etup Guidelines Load a maximum of 48 samples per OpenArray Digital PCR Plate Note Loading one sample per subarray does not provide sufficient confidence intervals for quantitative digital PCR however the single replicate setup can be used in experiments that screen candidate assays or optimal dilutions e Use a minimum of 64 technical replicates 1 subarray for each gDNA cDNA sample Apply sample names and assay labels to the digital PCR experiment either Prior to the run using the OpenArray System Software or After the run using the OpenArray Digital PCR Software Apply the identical sample name to the subarrays of each group of technical replicates The OpenArray Digital PCR Software combines data of replicate subarrays only if they share the same sample name If the replicate wells are named differently for example smpl012a and smpl012b the software analyzes the wells as different samples Apply unique assay names to the subarrays of plates that contain multiple TaqMan assays When a plate contains more than one assay label the wells according to the assay s that they contain The OpenArray Digital PCR Software can separate the data from multiple assays only if the associated wells are labeled with unique assay names No template We strongly recommend that you include at least one no template control NTC on controls each OpenArray Digital PCR Plate NTCs serve as negative controls that can be useful
53. eview and omit outliers 2 0 00 eee eee eee 36 Optimize the analysis settings 0 6 cece cece eee ee 38 Save and export the results 0 eee eee 39 Applied Biosystems OpenArray Real Time PCR System Digital PCR User Guide 27 3 Chapter 3 Analyze the Results Open the experiment Open the experiment Begin the analysis by opening the file exported by the OpenArray9 Real Time PCR System Software Guidelines The OpenArray Digital PCR Software can open and analyze Open the 1 experiment file 28 Exported raw amplification curve data csv files from digital PCR experiments run on an OpenArray System Results files saved by the OpenArray Digital PCR Software In the desktop either Double click wy OpenArray Digital PCR Software or e Select Start All Programs OpenArray Digital PCR OpenArray Digital PCR Software In the OpenArray Digital PCR Software window select File Load Data File In the Select data file dialog box select the raw amplification curve data csv file then click OK The software opens the selected experiment file analyzes the data using the default analysis settings and displays the results of the analysis Applied Biosystems OpenArray Real Time PCR System Digital PCR User Guide Chapter3 Analyze the Results Open the experiment The software displays the results of the analyzed experiment in the tabs and tables of the main window The elements in the following fi
54. experiments 10 Note See Appendix C on page 57 for a detailed explanation of 5 nuclease assays PCR and the analysis algorithm that is used by the OpenArray Digital PCR Software What is a digital PCR experiment Digital PCR is a biochemical technique used to quantify the number of starting copies of a target nucleic acid sequence in a genomic or complementary DNA sample Digital PCR experiments include the following components e Sample The genomic or complementary DNA sample that contains an unknown number of copies of the target nucleic acid sequence In a digital PCR experiment samples are diluted down to a limiting quantity such that most individual PCR reactions contain either zero or one target molecules Note Digital PCR experiments can be performed without knowing that most wells have either zero or one target molecules provided that some reactions within a sample group will have 0 copies e TaqMan OpenArray Digital PCR Master Mix An optimized mixture of dNTP salt buffer AmpliTaq DNA Polymerase and ROX dye passive reference designed for use with TaqMan assays and the OpenArray Real Time PCR System TaqMan Assay Includes forward and reverse primers and a specific fluorescent dye labeled probe for the target nucleic acid sequence The probe contains AFAM reporter dye linked to the 5 end of the probe A minor groove binder MGB at the 3 end of the probe MGBs increase the melting temper
55. f this product as unsorted municipal waste Follow local municipal waste ordinances for proper disposal provisions to reduce the environmental impact of waste electrical and electronic equipment WEEE Call your local Applied Biosystems Customer Service office for equipment pick up and recycling See www appliedbiosystems com for a list of customer service offices in the European Union T _o6 European Union customers RE General instrument safety Moving and lifting the instrument Moving and lifting stand alone computers and monitors Operating the instrument WARNING PHYSICAL INJURY HAZARD Use this product only as specified in this document Using this instrument in a manner not specified by Applied Biosystems may result in personal injury or damage to the instrument CAUTION PHYSICAL INJURY HAZARD The instrument is to be moved and positioned only by the personnel or vendor specified in the applicable site preparation guide If you decide to lift or move the instrument after it has been installed do not attempt to lift or move the instrument without the assistance of others the use of appropriate moving equipment and proper lifting techniques Improper lifting can cause painful and permanent back injury Depending on the weight moving or lifting an instrument may require two or more persons WARNING Do not attempt to lift or move the computer or the monitor without the assistance of others Depending on the
56. gure are described below About the interface OpenArray Plate Serial Number jESv43 v Heat Map Results f Display Results by Subarray Details Sample Group FS ID Sample Assay Dilution ELS A2 Normal SRY 0 25 SRY 0 50 SRY SRY SRY seP eP laseP RNaseP RNaseP RNaseP SRY SRY SRY SRY SRY Export Results Confidence Interval Range Lower Upper Confidence Confidence Level Level 0 6410 0 7425 1 3079 1 4733 0 7216 0 8309 2 3201 2 5915 Total Total Negatives Omitted Total Replicates Average Copies Per Reaction 0 6899 1 3881 0 7744 2 4520 Group A3 Normal A4 Normal A5 Normal A6 Normal A7 Normal A8 Mormal A9 Normal A10 Normal All Normal A12 Normal B1 Normal B2 Normal B3 Normal B4 Normal B5 Normal 0 6410 1 3079 0 7216 2 3201 0 7425 1 4733 0 8309 2 5915 Normal SRY Normal RNaseP Raji SRY Raji RNaseP Subarray Through Hole Details ct Ct Confidence v 0 0000 23 1437 E 0 0000 0 0000 Bar Plot of Copies Per Reaction 0 0000 37 4969 20 0 0000 24 3624 0 0000 0 0000 0 0000 27 6090 0 0000 30 3842 0 0000 0 0000 0 0000 0 0000 0 0000 0 0000 0 0000 0 0000 0 0000 0 0000 Annotation On Legend Displayed Graph Palette EH 2 Eu 103 Average Copies Per Reaction v 20100831 raw AC csv OpenArray 9 Digital PCR Software I10R3 Elements of the OpenArray Digital PCR Soft
57. h from manual to automatic baselining or move the baseline stop value to a lower Cy 2 cycles before the amplification curve for the sample crosses the threshold An amplification signal is detected in the early cycles no baseline can be set because the signal is detected too early Dilute the sample to increase the Cy value Follow the recommended sample preparation procedures for digital PCR see Prepare the DNA samples on page 18 Amplification curve shows a rising baseline Primer and probe interaction Adjust the threshold manually Select another assay from the same gene Amplification curve shows low ROX dye passive reference dye Inaccurate pipetting Little or no TaqMan OpenArray9 Digital PCR Master Mix Follow accurate pipetting practices Applied Biosystems OpenArray9 Real Time PCR System Digital PCR User Guide 41 Chapter 4 Troubleshooting Observation Possible cause Recommended solution More than the expected number of samples failed to properly amplify Degraded DNA Degraded DNA may not amplify as efficiently as high quality DNA so fluorescence intensities vary Perform a gel analysis to visualize the DNA quality Re extract samples that are degraded or remove them from the analysis See Prepare the DNA samples on page 18 Genomic DNA is not properly quantitated Samples with differing concentrations result in varied fluorescence
58. he probe is intact With TaqMan reagents a nonfluorescent quencher minor groove binder NFQ MGB can be used as the quencher With SYBR Green reagents no probe and therefore no quencher is used A solution that contains all components to run the PCR reaction except for the template sample standard or control Also called a PCR cocktail The PCR reaction components used to amplify the target and to detect amplification Process of collecting fluorescence data during PCR Data from the real time PCR are used to calculate results for quantification experiments Total number of identical reactions containing identical components and identical volumes A fluorescent dye used to detect amplification With TaqMan reagents the reporter dye is attached to the 5 end An oligonucleotide that flanks the 3 end of the amplicon The reverse primer and the forward primer are used together in PCR reactions to amplify the target Applied Biosystems OpenArray Real Time PCR System Digital PCR User Guide 75 Glossary reverse transcriptase ROX dye run method sample sample definition file sample target reaction stage step TaqMan reagents target technical replicates template threshold threshold cycle C7 76 An enzyme that converts RNA to cDNA A dye supplied by Applied Biosystems and precalibrated on the instrument ROX dye is used as the passive reference Definition of the react
59. idence Confidence Replicates Negatives Omitted Range E Reaction Level Level Mormal SRY 0 25000000 0 2173 0 1851 0 2551 768 618 0 0 1851 0 2551 Normal_SR 0 50000000 0 3017 0 2625 0 3467 768 568 0 0 2625 0 3467 Normal_SRY 1 00000000 0 6854 0 6187 0 7593 768 387 0 0 6187 0 7593 Normal RNaseP 0 25000000 0 3467 0 3040 0 3953 768 543 0 0 3040 0 3953 Normal RNaseP 0 50000000 0 7276 0 6580 0 8046 768 371 0 0 6580 0 8046 Normal_RNaseP 1 00000000 1 3405 1 2268 1 4647 768 201 0 1 2268 1 4647 Raji SRY 0 25000000 0 2012 0 1705 0 2376 768 628 0 0 1705 0 2376 Raji_SRY 0 50000000 0 4016 0 3548 0 4545 768 514 0 0 3548 0 4545 Raji SRY 1 00000000 0 7466 0 6757 0 8250 768 364 0 0 6757 0 8250 Raji_RNaseP 0 25000000 0 6449 0 5809 0 7158 768 403 0 0 5809 0 7158 Raji_RNaseP 0 50000000 1 2014 1 0982 1 3142 768 231 0 1 0982 1 3142 Raji_RNaseP 1 00000000 2 4097 2 1948 2 6456 768 69 0 2 1948 2 6456 w 1 Display Results By drop down list Select an option 3 Results table continued to display the associated data in the table and plot where Total Replicates The total number of through hole choices include reactions for the sample or replicate group that the software used in the analysis Total Negatives The total number of through hole reactions for the sample or replicate group that received negative calls 0 by the software Replicate Group Displays the analyzed data grouped by technical replicate group e
60. ion volume and the thermal profile for the instrument run The run method specifies the temperature time ramp and data collection points for all steps and stages of the instrument run The biological tissue or specimen that you are testing for a target gene A tab delimited text file txt that contains the following setup information well number sample name and custom sample properties The combination of the sample to test and the target to detect and quantify in one PCR reaction In the thermal profile a group of one or more steps Examples PCR stage cycling stage also called amplification stage and hold stage A component of the thermal profile For each step in the thermal profile you can set the ramp rate hold temperature and hold time duration You can turn data collection on or off for the ramp or the hold parts of the step PCR reaction components that consist of primers designed to amplify the target and a TaqMan probe designed to detect amplification of the target The nucleic acid sequence to amplify and detect Wells containing identical reaction components including sample which are important for evaluating precision The type of nucleic acid to add to the PCR reaction In amplification plots the level of fluorescence above the baseline and within the exponential growth region The threshold can be determined automatically or can be set manually The PCR cycle number at which the fluorescence meet
61. ission Editor Remove outliers 1 2 In the OpenArray Plate Omission Editor dialog box select the serial number of the plate that you want to modify from the OpenArray Plate Serial Number drop down list 3 In the plate grid select the through hole positions in the OpenArray Digital PCR Plate that you want to omit Note Right click a subarray and select an option to omit multiple through hole positions or to propagate an omission throughout the OpenArray Digital PCR Plate 4 Repeat steps 2 and 3 as needed to omit through hole positions from the OpenArray Digital PCR Plates 9 When you are finished omitting through hole positions click Accept Changes to apply the changes you have made Applied Biosystems OpenArray9 Real Time PCR System Digital PCR User Guide 37 3 Chapter3 Analyze the Results Optimize the analysis settings Optimize the analysis settings After you review the results of the default analysis you can optimize the analysis by modifying the analysis settings About the analysis The Analysis Settings dialog box allows you to change several analysis parameters settings including the way that the OpenArray Digital PCR Software assigns positive and negative calls during the analysis and the confidence interval that it uses The elements in the following figure are described below C OpenArray Digital PCR Analysis Settings Digital Analysis Settings Ct Confidence vs Ct Calculation Type ona Posit
62. ives Default 4000 m Negatives 3000 Y o o 1 Ct Confidence Analysis Settings Confidence Interval 1 Calculation Type drop down list Select the 2 Ct Confidence vs Ct Plot A plot contrasting the Cys method that the software will use to calculate positive and corresponding Cy confidence calculated for all negative calls for the through hole reactions of the through hole reactions on the loaded OpenArray Digital OpenArray Digital PCR Plates PCR Plate The plot displays a data point for each through hole reaction included in the analysis The OpenArray9 Digital PCR Software displays the data points in different colors to indicate the associated calls positive or negative Default The software calculates calls using a proprietary Life Technologies Corporation algorithm e Ct Range The software calculates calls using a discrete range of PCR cycles The software assigns 3 Confidence Interval field Select the confidence positive calls to all through hole reactions that yield interval that the OpenArray Digital PCR Software will use threshold cycles C4 that are within the range defined in to calculate the confidence interval for the copy number the Minimum Ct and Maximum Ct fields calculations e Ct Confidence Range The software calculates calls 4 Accept Closes the dialog box and reanalyzes the using a user defined minimum limit
63. lant If your sample DNA is extracted from blood do not use Heparin as an anti coagulant as it can inhibit PCR Use EDTA as an alternative Samples failed to amplify on the OpenArray System Contact Life Technologies Technical Support Perform proper bioinformatics on the sequence evaluate the assay design and consider redesigning the assay Verify the presence of the outlier Examine the performance of the sample in other assays to rule out problems caused by this particular sample such as sample impurity or degradation Search the public databases to see if the additional copies have been discovered Perform comparative sequencing on the subjects to identify any undocumented copies present under the primer or probe The reaction may not have enough copies of the target gene Sample is too dilute Perform a dilution series of the sample by increasing the quantity of input DNA added to the first point of the series Amplification occurs in the no RT controls gDNA contamination Perform bioinformatics Design the assay to span an exon exon junction Refer to the Custom TaqMan Genomics Assays Protocol Submission Guidelines PN 4367671 Bioinformatic Evaluation of a Sequence for Custom TaqMan Gene Expression Assays Tutorial from www appliedbiosystems com mprove sample extraction methods to eliminate gDNA See Prepare the DNA samples on page 18 Treat the sample with DNase
64. late File 3 Select the directory to receive the plate file a In the Plate file Creation dialog box click b In the Select the folder dialog box navigate to the directory that will receive the plate file then click Current Folder 4 In the New Serial Number field enter the serial number of the OpenArray Digital PCR Plate for which you are creating the plate setup file Plate File Creation File Destination ay New Serial Number asdfasdFasdfasdF Create File 5 Click Create File then click OK when prompted 6 Repeat steps 4 and 5 to create plate setup files for additional plates 7 Click Close The plate setup files created by the OpenArray Digital PCR Software can be used immediately after they are created You can add sample and assay information to the resulting experiment files following the PCR 8 Copy the files to the following folder on the OpenArray System computer lt drive gt Program Files BioTrove V PLATEFILES where drive is the computer drive on which the OpenArray software is installed The default installation drive is the C drive The OpenArray software must be able to access the plate setup file for each OpenArray Digital PCR Plate before the OpenArray System can perform real time cycling Applied Biosystems OpenArray9 Real Time PCR System Digital PCR User Guide Chapter 2 Prepare and Perform the Digital PCR Experiments 2 Perform the run Perform th
65. mples onto an OpenArray Digital PCR Plate e OpenArray Case Sealing Station Seals the OpenArray Digital PCR Plate Cases e OpenArray Real Time PCR System Performs thermal cycling and imaging of the experiment plates Computer Connects to the OpenArray Real Time PCR System About data collection The OpenArray Real Time PCR System collects raw fluorescence data after thermal cycling PCR amplification has been performed A data collection point data point on the OpenArray System consists of three phases 1 Excitation The OpenArray instrument illuminates all through holes of the experiment plate exciting the fluorophores in each reaction 2 Emission The OpenArray instrument optics collect the residual fluorescence emitted from the through holes of the experiment plate The resulting image consists only of light that corresponds to the range of emission wavelengths 3 Collection The OpenArray instrument assembles a digital representation of the residual fluorescence collected over a fixed time interval then stores the raw fluorescence image for analysis After a run the OpenArray software uses regions of interest ROI optical dye and background calibration data to determine the location and intensity of the fluorescence signals in each read the dye associated with each fluorescence signal and the significance of the signal Applied Biosystems OpenArray9 Real Time PCR System Digital PCR User Guid
66. n endogenous control If an inhibitor is present low concentrations yield higher than expected Cy values High concentration means more inhibition because the sample is not diluted c Rerun the assay with purified template Improve sample integrity extraction methods See Prepare the DNA samples on page 18 Poor reverse transcription RT conversion to cDNA Check the RNA sample for degradation e Input RNA could be too concentrated or too dilute Verify the concentration by optical density OD make new serial dilutions of template RNA from original stock then repeat the RT PCR Ensure that the RT PCR setup is performed under the appropriate conditions to avoid premature cDNA synthesis Check the RT reagents for contamination and or degradation Primer dimer formation and residual polymerase activity For optimal results run the reaction plate as soon as possible after completing the reaction setup If you cannot run a reaction plate within 2 hours after completing the reaction setup refrigerate or freeze the reaction plate until you can run it 44 Applied Biosystems OpenArray9 Real Time PCR System Digital PCR User Guide Chapter 4 Troubleshooting Observation Possible cause Recommended solution Amplification curve shows no amplification of the sample Cz 40 across all assays or in an unusually large number of assays One or more reaction components were not add
67. ndicates salts solvents and alcohols may be present Evaluate the current DNA extraction method and consider an alternative protocol Noisy signal above the threshold Empty through hole due to inaccurate loading e Visually inspect the array for the empty through hole e Pipette more than 5 uL of sample and reaction mix to the OpenArray 384 Well Sample Plate when loading 42 Applied Biosystems OpenArray Real Time PCR System Digital PCR User Guide Chapter 4 Troubleshooting Observation Possible cause Recommended solution All samples failed to amplify Numerous problems can cause mentioned issues in this table complete failure of an assay In addition to the previously consider the following A phenol chloroform DNA extraction method was used Use molecular biology grade phenol chloroform and remove all traces of phenol e Consider a bead based or column based extraction method The DNA sample contains impurities Dilute the DNA sample 1 10 to dilute impurities The DNA sample was not properly prepared Use an Applied Biosystems TaqMan Control Genomic DNA PN 4312660 to determine if the problem arises from the sample preparation Lower grade reagents were used Lower grade reagents may contain PCR inhibitors Use molecular biology grade reagents in all assay related experiments including DNA preparation Heparin was used as an anti coagu
68. o appear next to DANGERS WARNINGS and CAUTIONS that occur in the text of this and other product support documents Symbol Description Description ZN Indicates that you should proceed with appropriate caution and consult the product insert for further information If a product insert does not exist or if the product insert does not contain the symbol or the required information consult the user manual Indicates the presence of a pinching hazard and to proceed with appropriate caution Indicates the presence of moving parts and to proceed with appropriate caution Indicates the presence of a biological hazard and to proceed with appropriate caution Indicates the presence of an electrical shock hazard and to proceed with appropriate caution Indicates the presence of a laser light in the instrument and to proceed with appropriate caution AN AN Indicates the presence of a hot surface or other high temperature hazard and to proceed with appropriate caution gt pebei Indicates the presence of an ultraviolet light and to proceed with appropriate caution The following symbol applies to all Applied Biosystems electrical and electronic products placed on the European market after August 13 2005 Applied Biosystems OpenArray9 Real Time PCR System Digital PCR User Guide Appendix D Safety General instrument safety Symbol Description Do not dispose o
69. ohazard WARNING BIOHAZARD Biological samples such as tissues body fluids infectious agents and blood of humans and other animals have the potential to transmit infectious diseases Follow all applicable local state provincial and or national regulations Wear appropriate protective equipment which includes but is not limited to protective eyewear face shield clothing lab coat and gloves All work should be conducted in properly equipped facilities using the appropriate safety equipment for example physical containment devices Individuals should be trained according to applicable regulatory and company institution requirements before working with potentially infectious materials Read and follow the applicable guidelines and or regulatory requirements in the following In the U S U S Department of Health and Human Services guidelines published in Biosafety in Microbiological and Biomedical Laboratories www cdc gov biosafety publications index htm Occupational Safety and Health Standards Bloodborne Pathogens 29 CFR 1910 1030 www access gpo gov nara cfr waisidx 01 29cfr1910a_01 html Your company s institution s Biosafety Program protocols for working with handling potentially infectious materials Additional information about biohazard guidelines is available at www cdc gov In the EU Check local guidelines and legislation on biohazard and biosafety precaution and refer to the best practices published in
70. on in polymerase chain reactions Gene 93 125 128 Applied Biosystems OpenArray Real Time PCR System Digital PCR User Guide 71 Bibliography 72 Applied Biosystems OpenArray9 Real Time PCR System Digital PCR User Guide allele amplicon amplification amplification stage assay Assay ID assay information file AIF baseline chemistry Cr C1 algorithm Glossary In a diploid organism one of two DNA sequences found at the same locus for example a particular gene but located on homologous chromosomes Two corresponding alleles may have the identical sequence or they may differ somewhat often at one or more single base sites SNPs A segment of DNA amplified during PCR Part of the instrument run in which PCR amplifies the target Fluorescence data collected during amplification are displayed in an amplification plot and the data are used to calculate results Note Only quantitative real time PCR experiments not end point experiments take amplification data into account Part of the instrument run in which PCR amplifies the target The amplification stage called a cycling stage in the thermal profile consists of denaturing primer annealing and extension steps that are repeated Fluorescence data collected during the extension stage are displayed in an amplification plot and the data are used to calculate results With TagMan chemistry the last two steps of a PCR stage are typically combined See
71. onal Instruments Software Doing so renders the OpenArray Digital PCR Software inoperable If the National Instruments Software has been accidentally removed reinstall the OpenArray Digital PCR Software 2 Close all open applications 3 Start the OpenArray Digital PCR Software Installer Either Double click da OpenArray Digital PCR Software Installer or Load the OpenArray Digital PCR Software CD into the computer 4 Install the OpenArray Digital PCR Software as instructed IMPORTANT When prompted you must accept the licensing terms for both the OpenArray Digital PCR Software and the National Instruments Software 5 When the installation is complete click Finish 12 Applied Biosystems OpenArray9 Real Time PCR System Digital PCR User Guide Materials Order an assay Storage Safety data sheets Compatible reagents Chapter 1 Overview 1 Materials For details on how to order a TaqMan assay refer to the TaqMan assays products page at www allgenes com the TagMan Gene Expression Assays Protocol PN 4333458 or the TagMan Copy Number Assays Protocol PN 4397425 Part number Part Storage conditions 4458071 OpenArray Digital PCR Plates 10 pack 15 to 25 C 4460694 OpenArray Digital PCR Plates 3 pack 4458080 TaqMan OpenArray Digital PCR Master Mix 5mL 15 to 25 C until first 4458086 TaqMan OpenArray Digital PCR Master Mix 1 5 mL u
72. ontainer storage Minimize contact with chemicals Wear appropriate personal protective equipment when handling chemicals for example safety glasses gloves or protective clothing For additional safety guidelines consult the SDS Minimize the inhalation of chemicals Do not leave chemical containers open Use only with adequate ventilation for example fume hood For additional safety guidelines consult the SDS Handle chemical wastes in a fume hood After emptying a waste container seal it with the cap provided Dispose of the contents of the waste tray and waste bottle in accordance with good laboratory practices and local state provincial or national environmental and health regulations Waste disposal If potentially hazardous waste is generated when you operate the instrument you must Characterize by analysis if necessary the waste generated by the particular applications reagents and substrates used in your laboratory Ensure the health and safety of all personnel in your laboratory Ensure that the instrument waste is stored transferred transported and disposed of according to all local state provincial and or national regulations IMPORTANT Radioactive or biohazardous materials may require special handling and disposal limitations may apply Applied Biosystems OpenArray Real Time PCR System Digital PCR User Guide 69 Appendix D Safety Biological hazard safety Biological hazard safety General bi
73. ow Note For information on loading plates refer to the TaqMan OpenArray Real Time PCR Plates Protocol PN 4458840 or the OpenArray AccuFill System User Guide PN 4456986 For information on performing real time imaging and exporting data refer to the OpenArray Real Time PCR System User Guide PN 4458837 Prepare the digital PCR reactions 1 Prepare the reaction mix 2 Load the reaction mix and samples into the OpenArray9 Sample Plate l Load the OpenArray Digital PCR Plate OpenArray AutoLoader Procedure OpenArray AccuFill System 1 Prepare for loading Procedure 2 Place a OpenArray Digital PCR Plate in 1 Prepare for loading an OpenArray AutoLoader Plate Holder 2 Place an OpenArray Digital PCR Plate lt 1 17M 3 Load the OpenArray AutoLoader Tip fto tne OpenAnay SAGOUESTE syster Blocks Load the OpenArray AccuFill Tips Run the OpenArray AccuFill System Seal the OpenArray Digital PCR Plate Perform thermal cycling 4 Run the OpenArray AutoLoader 5 Seal the OpenArray Digital PCR Plate 6 Perform thermal cycling l l Perform real time imaging o oO P OQ Create the plate setup file tpf using the OpenArray Digital PCR Software Set up the OpenArray Real Time qPCR Analysis Software Enter sample information in the OpenArray software Place the loaded OpenArray Digital PCR Plates into the OpenArray Real Time PCR System
74. patibility standards Safety and electromagnetic compatibility standards This section provides the following information on e U S and Canadian safety standards Canadian EMC standard European safety and EMC standards Australia and New Zealand EMC standards U S and Canadian The OpenArray AccuFill System OpenArray Case Sealing Station and safety standards OpenArray Instrument have been tested to and comply with the standards U UL 61010 1 2nd Edition CSA C22 2 No 61010 1 Safety Requirements for Electrical CV S L US Equipment for Measurement Control and Laboratory Use Part 1 General Requirements Canadian EMC The OpenArray AccuFill System OpenArray Case Sealing Station and standard OpenArray Instrument have been tested to and comply with ICES 001 Issue 3 Industrial Scientific and Medical Radio Frequency Generators European safety Safety and EMC The OpenArray AccuFill System OpenArray Case Sealing Station and standards OpenArray Instrument meet European requirements for safety Low Voltage Directive 2006 95 EC This instrument has been tested to and complies with standards EN 61010 1 2001 Safety Requirements for Electrical Equipment for Measurement Control and Laboratory Use Part 1 General Requirements The OpenArray Instrument has been tested to and complies with the standard EN 60825 1 Radiation Safety of Laser Products Equipment Classification Requirements and
75. r Guide 11 1 Chapter 1 Overview Install the software Install the software System Computer Monitor Operating system requirements e Pentium 4 or compatible processor e 1280x 1024 pixel Microsoft Windows XP 1 0 GHz resolution for full Service Pack 2 or later e 1GB of RAM screen display e 22 MB disk space e 16 inch or larger e UL listed e 32 bit color e UL listed e CD drive if installing from a CD Browser with a internet connection if installing from the internet Install the IMPORTANT To install the software your user account must have administrative Open Array Digital privileges to the Microsoft Windows operating system PCR Software 1 Download the OpenArray Digital PCR Software a Go to www appliedbiosystems com b In the Home page of the Applied Biosystems web site click Support c In the Support page select OpenArray Digital PCR Software in the Software Downloads Patches amp Updates drop down list then click Continue d Inthe OpenArray Digital PCR Software page click Download Free Software e Complete the registration as directed by the web site then follow the instructions to download and install the OpenArray Digital PCR Software Note The OpenArray Digital PCR Software Installer also installs the National Instruments LabVIEW Runtime Engine a critical component of the OpenArray Digital PCR Software IMPORTANT Do not uninstall the Nati
76. ransforms the signal to electronic data and saves the data in the experiment file A reagent used to dilute a sample or standard before it is added to the PCR reaction A software feature that allows you to export experiment setup files experiment results instrument information and security and auditing settings to spreadsheet presentation or text files You can edit the default location of the exported file Oligonucleotide that flanks the 5 end of the amplicon The reverse primer and the forward primer are used together in PCR reactions to amplify the target In the thermal profile the stage that holds the temperature constant for a defined period of time A stage that includes one or more steps You can add a holding stage to the thermal profile to activate enzymes to inactivate enzymes or to incubate a reaction A software feature that allows you to import plate setup information or security settings before an experiment run You can also import information into some libraries in the system An analysis setting for the Baseline Threshold algorithm You enter the baseline start and end cycles for the amplification plot See also baseline An analysis setting for the Baseline Threshold algorithm You enter the threshold value and select whether to use automatic baseline or manual baseline values The software uses the baseline and the threshold values to calculate the threshold cycle C7 A plot of the complete spectral con
77. res as recommended in the SDS Comply with all local state provincial or national laws and regulations related to chemical storage handling and disposal Applied Biosystems OpenArray9 Real Time PCR System Digital PCR User Guide 67 Appendix D Safety SDSs SDSs About SDSs Chemical manufacturers supply current Safety Data Sheets SDSs with shipments of hazardous chemicals to new customers They also provide SDSs with the first shipment of a hazardous chemical to a customer after an SDS has been updated SDSs provide the safety information you need to store handle transport and dispose of the chemicals safely Each time you receive a new SDS packaged with a hazardous chemical be sure to replace the appropriate SDS in your files Obtaining SDSs The SDS for any chemical supplied by Applied Biosystems is available to you free 24 hours a day To obtain SDSs 1 Goto www appliedbiosystems com click Support then select SDS 2 In the Keyword Search field enter the chemical name product name SDS part number or other information that appears in the SDS of interest Select the language of your choice then click Search 3 Find the document of interest right click the document title then select any of the following Open To view the document Print Target To print the document Save Target As To download a PDF version of the document to a destination that you choose Note For the SDSs of chemicals not distribute
78. resistant pipette tips Clean lab benches and equipment periodically with 10 bleach solution Clean the OpenArray Autoloader accessories After each use clean the following OpenArray Autoloader accessories e OpenArray Plate Guide Set e OpenArray Autoloader Tip Block e OpenArray Autoloader Plate Holder For cleaning procedures see the TaqMan OpenArray Real Time PCR Plates Protocol PN 4458840 Applied Biosystems OpenArray Real Time PCR System Digital PCR User Guide 47 48 Appendix A Prevent Contamination Clean the OpenArray Autoloader accessories Applied Biosystems OpenArray Real Time PCR System Digital PCR User Guide This appendix includes About the OpenArray plates Digital PCR experiment setup Example plate layout 1 Example plate layout 2 Applied Biosystems OpenArray Real Time PCR System Digital PCR User Guide APPENDIX B Example Layouts 49 Appendix B Example Layouts About the OpenArray plates About the OpenArray plates OpenArray 384 Well Sample Plates OpenArray Digital PCR Plates 50 The OpenArray 384 Well Sample Plate is a 384 well microtiter plate You combine the TaqMan OpenArray Digital PCR Master Mix DNA samples and TaqMan Assays in the sample plate then use the OpenArray AccuFill System or the OpenArray AutoLoader to transfer the mixture from the sample plate to an OpenArray Digital PCR Plate Th
79. riments 2 Prepare the DNA samples Determine the In a digital PCR experiment performed on an OpenArray System gDNA or cDNA optimal dilution samples are diluted down to a limiting quantity such that most individual PCR reactions in the through holes of each subarray contain either zero or one target molecules The procedure for determining the optimal dilution for a sample differs depending on whether or not the target copy number per genome of the sample is known If the target copy number per genome of your samples is known dilute the samples so that when aliquotted to a subarray each through hole reaction will contain approximately 0 6 to 1 6 copies of the target sequence For example assuming 3 3 pg copy of a given gene are present per genome and a 33 nL through hole volume the stock gDNA in a given sample would be diluted down to 60 pg uL 0 06 ng uL in the final reaction to give 0 6 copies per through hole Copies hole Copies pL ng pL 0 6 18 18 0 06 If the target copy number per genome is unknown we recommend that you determine the optimal dilution by loading a OpenArray Digital PCR Plate with a three or four fold dilution series of the each sample at the expected digital range By assaying three to four data points above and below the expected digital range you ensure that one of the data points is within the optimal digital range How to determine the target copy number per genome To help determine copy n
80. rray Digital PCR Plates 0 eee 21 Create the plate setup files lesse 22 Before you perform digital PCR experiments on the OpenArray Real Time PCR System you must choose an experiment layout for your OpenArray Digital PCR Plates The OpenArray Digital PCR Software does not restrict the placement of TaqMan Assays and samples on the OpenArray Digital PCR Plates The number and arrangement of TaqMan assays and samples that you can load in a OpenArray Real Time PCR System can vary based on the experiment layout that you select For more information on choosing a layout see Appendix B Example Layouts on page 49 For the following hazard see the complete safety alert description in Appendix D Safety on page 61 CAUTION CHEMICAL HAZARD TaqMan OpenArray Digital PCR A Master Mix 2X and TaqMan Assay 1 Remove the following from the freezer and allow them to thaw at room temperature e TaqMan OpenArray Digital PCR Master Mix e TaqMan Assay s 2 Vortex then centrifuge the DNA samples for 1 minute at 1000 rpm 3 Review the concentration of your genomic DNA samples then prepare a dilution of stock gDNA 0 15 ng uL Material Volume pL Stock gDNA 10ng pL 15 TE Buffer 1X 485 Total 500 See Quantity of DNA on page 18 for information on the recommended starting concentration for g DNA samples 4 Gently invert the tube of TaqMan OpenArray Digital PC
81. s the threshold in the amplification plot Applied Biosystems OpenArray9 Real Time PCR System Digital PCR User Guide Part Number 4459761 Rev A 10 2010 gt applied A biosystems by Lefe technologies Headquarters 5791 Van Allen Way Carlsbad CA 92008 USA Phone 760 603 7200 www lifetechnologies com 445976148 Technical Resources and Support For the latest technical resources and support information for all locations please refer to our Web site at www appliedbiosystems com
82. se then 2 to 8 C Store the OpenArray and TaqMan materials and reagents according to the labels on the packaging For the SDS of any chemical not distributed by Applied Biosystems contact the chemical manufacturer Before handling any chemicals refer to the SDS provided by the manufacturer and observe all relevant precautions Note Where noted products are available from major laboratory suppliers MLS Table 1 OpenArray9 Sample Plate set up and loading Product LAM Source Corning9 96 Well Microplate Aluminum Sealing Tape 6570 Corning Life Nonsterile Sciences Finnpipette Multichannel Digital Pipettor 5 to 50 uL 4452470 OpenArray 384 Well Sample Plates 4406947 OpenArray AccuFill System 4457243 OpenArray AccuFill System Tips 1 tip 4457246 10 tips 4458107 OpenArray AutoLoader 4409360 OpenArray AutoLoader Plate Holder 20384 OpenArray AutoLoader Tip Block 20322 a OpenArray Case Sealing Station 4409361 OpenArray Digital PCR Plates 3 plate 4460694 10 plates 4458071 OpenArray Loader Tips 1 tips 4404571 10 tips 4404604 OpenArray Real Time PCR Accessories Kit 4453975 OpenArray Real Time PCR Plate Frame 3 Pack 4453942 13 Applied Biosystems OpenArray9 Real Time PCR System Digital PCR User Guide Chapter 1 Overview Materials Product al Source number
83. shold value to within the appropriate range Assay design or synthesis failure The wrong sequence was submitted to Applied Biosystems Verify that the sequence you submitted is correct Check for an alternative transcript or a splice variant Assay is designed in a variable region of the gene transcript Verify that the location targeted by the assay is not within the 5 untranslated region UTR which can be highly variable between transcripts If the assay is designed within the 5 UTR select a different assay that is within the coding region of the transcript Otherwise select an assay for an alternative transcript or splice variant cDNA conversion failed Check the RNA integrity and concentration Check for RNase activity Follow the recommended thermal profile Repeat the RT step using new reagents Applied Biosystems OpenArray Real Time PCR System Digital PCR User Guide 45 Chapter 4 Troubleshooting Observation Possible cause Recommended solution Amplification curve shows no amplification of the sample Cz 40 in the target assay One or more of the reaction components was not added Check your pipetting equipment and or technique Incorrect dye components were selected Check the settings of the dye components before data analysis The gene is not expressed in the tested sample e Verify by Rerunning the sample using the same assay
84. sults After completing the analysis you can save the data for later use by the OpenArray Digital PCR Software or for downstream analysis by a third party software Save modifications If you modified the layout of the samples assays and dilutions on the OpenArray to the original Digital PCR Plates in the OpenArray Digital PCR Software you can save the changes experiment file to the original experiment file by selecting File Save Data File in the main window Save the analysis To save the analyzed digital PCR experiment data for later use by the OpenArray Digital PCR Software 1 In the OpenArray Digital PCR Software window select File Save As 2 In the Save As dialog box configure the settings a Navigate to the directory that you want to receive the file b In the File name field enter a name for the analyzed file 3 Click Save Export the analysis To export the analyzed digital PCR experiment data as a Microsoft Excel xls file for downstream analysis by a third party software 1 In the OpenArray Digital PCR Software window click Export Results 2 In the Select File to Save Results dialog box configure the settings a Navigate to the directory that you want to receive the file b In the File name field enter a name for the exported file 3 Click Save Applied Biosystems OpenArray9 Real Time PCR System Digital PCR User Guide 39 3 Chapter 3 Analyze the Results Save and export the results 40
85. template was not optimal Examine the confidence values to assess the reliability of each result Change the plot scale In the Results tab of the OpenArray Digital PCR Software window right click the plot then select the desired setting Copy Data Copies the plot into the clipboard for transfer to another software such as a word processor a slide show application or a graphics editor e AutoScale Y Select to allow the software to automatically adjust the scale of the y axis e Mapping Style Select the desired scale of the plot Linear or Logarithmic Applied Biosystems OpenArray9 Real Time PCR System Digital PCR User Guide 33 Chapter 3 Analyze the Results View the data View the data After you load a raw amplification curve data csv file you can use the Heat Map tab to review the processed data as explained below About the Heat The Heat Map tab allows you to view the data of three OpenArray Digital PCR Plates as two dimensional heat maps where color is used to indicate numerical variation in the data matrix The elements in the following figure are described below Map tab Heat Map Results Data to Display ct v Graph Palette M Im 1 e ul m w ul 1 Data to Display drop down list Select an option to display the associated data in the heat map where choices include e Ct Displays through hole threshold cycle C4 data for the OpenArray Digital PCR Plate s where in
86. tensity is expressed in PCR cycle number e Amplification Displays positive negative call data for the OpenArray Digital PCR Plate s The plot is a simplified view of the Ct heat map where white indicates that a Cy was calculated for the associated through hole position and black indicates that no C7 was calculated Positions displayed in red indicate omitted through holes Omitted Displays through hole data omitted by the software for the OpenArray Digital PCR Plate s where red indicates an omitted position Plate Layout Displays sample assay and dilution settings for the subarrays of the OpenArray Digital PCR Plate s 2 4 Crosshair tool Select to change the mouse cursor to a crosshair which provides accurate selection of data 3 39 Magnifier tool Opens a menu of magnification tools for manipulating the heat map charts Zooms the heat map to the selected region Zooms the heat map horizontally to the selected region without expanding the vertical axis Zooms the heat map vertically to the selected region without expanding the horizontal axis Resets the view to display the entire heat map xe Zooms out the heat map incrementally with each click e Zooms in the heat map incrementally with each click 4 W Grab tool Select to change the mouse cursor to a hand which allows manual repositioning of the heat maps 5 Heat map Displays a heat map of the OpenArray9 Digital
87. th the grounding conductor disconnected WARNING ELECTRICAL HAZARD Use properly configured and approved line cords for the voltage supply in your facility WARNING ELECTRICAL HAZARD Plug the system into a properly grounded receptacle with adequate current capacity Overvoltage rating The OpenArray AccuFill System OpenArray Case Sealing Station and OpenArray Instrument have an installation overvoltage category of II and are classified as portable equipment Workstation safety Correct ergonomic configuration of your workstation can reduce or prevent effects such as fatigue pain and strain Minimize or eliminate these effects by configuring your workstation to promote neutral or relaxed working positions CAUTION MUSCULOSKELETAL AND REPETITIVE MOTION HAZARD These hazards are caused by potential risk factors that include but are not limited to repetitive motion awkward posture forceful exertion holding static unhealthy positions contact pressure and other workstation environmental factors To minimize musculoskeletal and repetitive motion risks e Use equipment that comfortably supports you in neutral working positions and allows adequate accessibility to the keyboard monitor and mouse Position the keyboard mouse and monitor to promote relaxed body and head postures Applied Biosystems OpenArray Real Time PCR System Digital PCR User Guide 65 Appendix D Safety Safety and electromagnetic com
88. to enter the serial numbers and place OpenArray Digital PCR Plates in Positions 2 and 3 IMPORTANT If you are running fewer than three OpenArray Digital PCR Plates use Position 1 for one plate and Positions 1 and 2 for two plates IMPORTANT Leave the Input Plate Serial Numbers dialog box open then proceed to Enter sample information on page 24 If you close the dialog box the information you have entered will be lost Applied Biosystems OpenArray Real Time PCR System Digital PCR User Guide 23 2 Chapter 2 Prepare and Perform the Digital PCR Experiments Perform the run Enter sample information Run the OpenArray Digital PCR Plate 24 Note Refer to the OpenArray Real Time PCR System User Guide PN 4458837 for more information on entering sample data using the OpenArray software Entering sample information allows you to Track the sample plates and map the sample plate areas to each OpenArray Digital PCR Plate Associate information about the samples with the results in order to analyze the data After loading the OpenArray Digital PCR Plates you can import sample information from a csv file or enter it manually You can enter sample information after a run has completed however we recommend that you enter the sample information before starting the run Note Refer to the OpenArray Real Time PCR System User Guide PN 4458837 for more information on operating the OpenArray Real Time PC
89. tocol PN 4458840 for information on loading and preparing the OpenArray Digital PCR Plate Applied Biosystems OpenArray Real Time PCR System Digital PCR User Guide 21 2 Chapter 2 Prepare and Perform the Digital PCR Experiments Prepare the digital PCR experiment Create the plate setup files 22 Before you can run the loaded OpenArray Digital PCR Plates you must create a plate setup file tpf for each plate that you intend to run The plate setup file describes the following aspects of an OpenArray Digital PCR Plate and the associated experiment e Imaging and thermal cycling protocol temperature and time parameters that define the PCR and the stages cycles designated for data collection e OpenArray Digital PCR Plate and OpenArray System identification information serial numbers and bar codes Plate setup files are created using the OpenArray Digital PCR Software which automatically populates each file with thermal cycling protocol and plate identification information Assay and sample information can be added to the digital PCR experiment Before you run the associated plate using the OpenArray System Software or e After the run using the OpenArray Digital PCR Software Create the file 1 In the desktop double click E4 OpenArray Software or select Start All Programs OpenArray Digital PCR OpenArray Digital PCR Software 2 In the OpenArray Digital PCR Software window select File New P
90. tribution of each dye for the selected well s over the duration of the PCR run The task for target assays in wells that contain water or buffer instead of sample No amplification of the target should occur in negative control wells Previously called no template control NTC See negative control NC Molecules that are attached to the 3 end of TaqMan probes When the probe is intact the nonfluorescent quencher NFQ prevents the reporter dye from emitting fluorescence signal Because the NFQ does not fluoresce it produces lower background signals resulting in improved precision in quantification The minor groove binder MGB increases the melting temperature Tm of the probe without increasing its length allowing for the design of shorter probes Applied Biosystems OpenArray9 Real Time PCR System Digital PCR User Guide omit through hole outlier passive reference plate layout plate setup file primer probe mix quantity quencher reaction mix reagents real time PCR replicates reporter reverse primer Glossary An action that you perform before reanalysis to omit one or more through holes from analysis Because no algorithms are applied to omitted through holes omitted through holes contain no results You can add through holes back in to the analysis no information is permanently discarded A measurement such as a Cr that deviates significantly from the measurement of the other replicates
91. ts 2 Perform the run Export the cycling Before you can analyze the data collected from OpenArray Digital PCR Plates you data must export the analyzed cycling data to a comma separated value csv file IMPORTANT The exported file must be an export of the project file that contains only the raw amplification curve data To export all columns from the Assays pane or Sample Data pane 1 Select File Export Cycling Data Note If you are prompted to save the data before exporting the data click OK 2 In the Export Cycling Data dialog box select Raw Amplification Curve and deselect all other options then click Export Export Cycling Data l Baselined Amplification Curve I dF dT Melting Curve M Raw Amplification Curve Raw Melting Curve l Summary Results Standard Curve 3 In the Save As dialog box browse to the desired location enter a file name then click Save Applied Biosystems OpenArray9 Real Time PCR System Digital PCR User Guide 25 2 Chapter 2 Prepare and Perform the Digital PCR Experiments Perform the run 26 Applied Biosystems OpenArray9 Real Time PCR System Digital PCR User Guide CHAPTER 3 Analyze the Results In this chapter Open the experimenta i22 tex a ne NN EEG E HU E EE RE 28 Edit the experiment information lissssssseeee ee 30 View the results ce seerm re Er ee d e ERR RR RARE UE wie PAR eH E unes 32 Maew the data 255 aso duties eet eR as airs en SU d eon stl e Do 34 R
92. umber per genome collect the following information 1 Ifthe source or species of the gDNA is known but the genome size of the organism of interest is unknown refer to http www cbs dtu dk databases DOGS index html to determine the size of the genome in question 2 Once the size of the genome is known determine the mass of the genome using the following formula m n 1 096 x 10 g bp where m is the genome mass in grams and n is the genome size in base pairs The following example calculates the mass of the human genome using the Celera Genomics estimate of 3 0 x 10 bp haploid m 3 0 x 10 bp 1 096 x 10 g bp m 3 3 x10 g or 33 pg The example is relevant to any gene that is present at the normal rate of two copies per diploid genome such as RNase P and provides a basis to perform a digital screening experiment to determine the optimal digital range Applied Biosystems OpenArray9 Real Time PCR System Digital PCR User Guide 19 2 Chapter 2 Prepare and Perform the Digital PCR Experiments Prepare the digital PCR experiment Prepare the digital PCR experiment Choose an experiment layout Prepare reaction mix and sample plate 20 Preparation of digital PCR experiments involves the following steps that must be completed prior to running the OpenArray Digital PCR Plates Choose an experiment layout 0 eee 20 Prepare reaction mix and sample plate llssseeeeleeeeeeee 20 Load the OpenA
93. ware include 1 Menu bar File menu Opens results files opens or saves an analysis or exits the software 5 Subarray Details table Displays the subarrays of the plate selected in the OpenArray Plate Serial Number drop down list Select a row to display the associated data in the Display menu Opens the Sample Assay Dilution Editor subartey Through note Deals table for editing the assay and dilution information for the experiment and opens the OpenArray Plate Omission Editor for omitting through hole data e ID The coordinates row and column of the subarray Sample The name of the sample loaded to the subarray Assay The name of the assay loaded to the subarray e Advanced menu Opens the analysis and plot settings e Dilution The dilution point of the sample loaded to the Help menu Displays information about the software subarray 6 Subarray Through Hole Details table Displays the data of the subarray selected in the Subarray Details table 2 OpenArray Plate Serial Number drop down list Select a barcode to display the associated plate data The list contains the barcodes of all plates included in the P experiment Note A check mark appears next to the displayed plate 3 Heat Map tab Displays heat maps of the analyzed and unprocessed data for the OpenArray plates loaded for analysis see page 34 for more information 4 a Results Tab Displays the analyzed dat
94. xample Layouts Example plate layout 2 Example plate layout 2 This section describes an example plate layout for a digital PCR experiment that evaluates a short dilution range of two samples for two nucleic acid targets Description In this example Dilutions of two samples S01 and S02 are evaluated for two nucleic acid targets using a pair of TaqMan assays A01 and A02 Atwo fold three point dilution series of each sample is evaluated for the nucleic acid targets The dilutions are such that each through hole reaction contains either a 0 5 1 0 or 2 0 dilution of the stock solution The samples are arrayed in replicate so that the plate evaluates each sample dilution assay combination in quadruplicate The plate contains a total of 12 replicate groups where each group consists of 256 replicate reactions 4 subarrays x 64 through holes 256 reactions Experiment layout The following figure illustrates the plate layout for the example digital PCR experiment 11 12 S02 S02 0 5 0 5 A01 A01 S02 S02 0 5 0 5 A01 A01 S02 S02 0 5 0 5 A02 A02 S02 S02 05 0 5 A02 A02 54 Applied Biosystems OpenArray9 Real Time PCR System Digital PCR User Guide Sample plate setup Subarray locations Appendix B Example Layouts Example plate layout 2 Sample dilutions and assays are loaded to the 384 well sample plate as follows
95. youts Example plate layout 1 Sample dilutions and assays are loaded to the 384 well sample plate as follows Sample Dilution Assay Load to wells Sample 1 1 0e4 Assay 1 A1 B1 C1 D1 Sample 1 1 0e3 Assay 1 A2 B2 C2 D2 Sample 1 100 Assay 1 A3 B3 C3 D3 Sample 1 10 Assay 1 A4 B4 C4 D4 Sample 1 1 0 Assay 1 Ab B5 C5 D5 Sample 1 0 1 Assay 1 A6 B6 C6 D Sample 2 1 0e4 Assay 1 A7 B7 C7 D7 Sample 2 1 0e3 Assay 1 A8 B8 C8 D8 Sample 2 100 Assay 1 A9 B9 C9 D9 Sample 2 10 Assay 1 A10 B10 C10 D10 Sample 2 1 0 Assay 1 A11 B11 C11 D11 Sample 2 0 1 Assay 1 A12 B12 C12 D12 Sample plate Plate area 1 Plate area 2 Yate area 4 eae q 1 te area 6 SSS 1 area 8 quee Y 4 Subarray A1 Te ESI ESTERI EIER E IERI EESTI t9 E E ES E cd ES E e E US E EE EE ES EE Ee EE d d Ee EE SET ES EE ESSERE 9 OpenArray plate SS Subarray D12 When you transfer the samples from the sample plate to the OpenArray Digital PCR Plate program the OpenArray Accufill System or the AutoLoader System to perform one load The system transfers the samples to the following locations of the OpenArray Digital PCR Plate Sample plate Load OpenArray Digital PCR Software subarray locations 1 1 Through holes A1 through H8 Applied Biosystems OpenArray9 Real Time PCR System Digital PCR User Guide 53 Appendix B E

OpenArray® Real-Time PCR System, Digital PCR Experiments

Contents

Download Pdf Manuals

Related Search

Related Contents

OpenArray&reg; Real-Time PCR System, Digital PCR Experiments

Contents

Download Pdf Manuals

Related Search

Related Contents

OpenArray® Real-Time PCR System, Digital PCR Experiments