Transcription
TaqMan Gene Expression Assays—TaqMan Array CardsUSER GUIDEPublication Number 4400263Revision GFor Research Use Only. Not for use in diagnostic procedures.
Life Technologies Corporation 6055 Sunol Blvd Pleasanton, California 94566 USAFor descriptions of symbols on product labels or product documents, go to thermofisher.com/symbols-definition.The information in this guide is subject to change without notice.DISCLAIMER: TO THE EXTENT ALLOWED BY LAW, THERMO FISHER SCIENTIFIC INC. AND/OR ITS AFFILIATE(S) WILL NOT BELIABLE FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE, OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH ORARISING FROM THIS DOCUMENT, INCLUDING YOUR USE OF IT.Revision history: Pub. No. 4400263RevisionGFEDCDateDescription8 March 2021 Added the QuantStudio 7 Pro Real-Time PCR System and the QuantStudio Design and Analysis Software v2. Updated centrifuges. Updated the name of the cloud-based platform to Thermo Fisher Connect andupdated the URLs. Corrected thermal cycling conditions for the following Master Mixes:20 November 201810 July 20185 March 2018July 2010 –TaqMan Fast Advanced Master Mix–TaqMan Gene Expression Master Mix–TaqMan Universal Master Mix II, with UNG–TaqMan Universal Master Mix II, no UNG Removed TaqMan Fast Universal PCR Master Mix, no AmpErase UNG as acompatible Master Mix. Updated algorithms for data analysis for 7900HT Fast Real-Time PCR Instrument. Updated recommended action if too much cDNA template is added. Added information about recommended algorithms for data analysis. Updated information to obtain files for the cards. Added new instruments, Master Mixes, and other applicable products. Added thermal cycling protocols for all compatible Master Mixes. Updated guidelines for reverse transcription. Removed content that is described in other resources; added references asappropriate. Updated for general style, formatting, and branding.Baseline for this revision history.Important Licensing Information: These products may be covered by one or more Limited Use Label Licenses. By use of theseproducts, you accept the terms and conditions of all applicable Limited Use Label Licenses.Trademarks: All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. TaqMan is aregistered trademark of Roche Molecular Systems, Inc., used under permission and license. Eppendorf and MixMate are trademarks ofEppendorf AG. 2021 Thermo Fisher Scientific Inc. All rights reserved.
Contents CHAPTER 1 Product information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5Product description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 Over of a TaqMan Array Card . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6Contents and storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 Order TaqMan Array Cards . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8Required materials and equipment not supplied . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8Workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 CHAPTER 2 Guidelines for preparation of cDNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12Guidelines for isolation of high–quality RNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12Guidelines for preparing cDNA templates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 CHAPTER 3 Perform real-time PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13Procedural guidelines for performing real-time PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13Guidelines for quantity of cDNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14Combine cDNA and Master Mix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 Prepare a TaqMan Array Card . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15Set up and run the real-time PCR instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15Analyze the results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16Algorithms for data analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 APPENDIX A Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19Troubleshooting: After removing the card from packaging . . . . . . . . . . . . . . . . . . . . . . . . . . . 19Troubleshooting: After loading PCR reaction mix into the card . . . . . . . . . . . . . . . . . . . . . . . 19Troubleshooting: After centrifuging the card . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20Troubleshooting: After running the card and reviewing run results . . . . . . . . . . . . . . . . . . . . 20TaqMan Gene Expression Assays User Guide—TaqMan Array Cards3
Contents APPENDIX B Detailed procedures for preparation of a TaqManArray Card . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25Guidelines for preparation of a card . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 TaqMan Array Card diagram . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26Load the PCR reaction mix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27Centrifuge the card . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27Seal the card . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 APPENDIX C Supplemental information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32Endogenous controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32Candidate endogenous controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32 TaqMan Gene Expression Assays chemistry overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 TaqMan MGB probes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33About the 5' nuclease assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34Instrument software and setup files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35How to import setup files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35Thermal protocols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36Best practices for PCR and RT-PCR experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Good laboratory practices for PCR and RT-PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Use UNG to prevent false-positive amplification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Detect fluorescent contaminants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38383839APPENDIX D Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40Chemical safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41Biological hazard safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42 APPENDIX E Documentation and support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43Related documentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43Customer and technical support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44Limited product warranty . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 444TaqMan Gene Expression Assays User Guide—TaqMan Array Cards
1Product information Product description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5Over of a TaqMan Array Card . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6Contents and storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7Order TaqMan Array Cards . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8Required materials and equipment not supplied . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8Workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11Product descriptionThe procedures in this document are for use with dried-down TaqMan Gene Expression Assays thatare configured on TaqMan Array Cards.TaqMan Gene Expression Assays are a comprehensive collection of predesigned, preformulatedprimer and probe sets to perform quantitative gene expression studies on various species.For a current list of available species and assays, use the assay search tool at thermofisher.com/taqmangeneexpression. TaqMan Gene Expression Assays– Are a general collection of assays that target protein‑coding transcripts from various speciesand for specific diseases, pathways, or biological processes.– Include TaqMan Non‑coding RNA Assays that target long non‑coding RNA (ncRNA) inhuman, mouse, and rat species. These assays are designed for ncRNAs that are 60 nt inlength. Endogenous control assays (see page 32 for more information)The following configurations are lated and predefined TaqMan GeneFixed-contentExpression Assays that are manufacturedTaqMan Array Cardsand stocked in advanceNoCards configured with a suggested selectionFlexible-contentof TaqMan Gene Expression Assays, TaqMan Array Cards categorized by disease, pathway, orbiological processPreselected assays can be substitutedwith other predesigned assays that targetTaqMan Gene Expression Assays moreapplicable to experiment needsCustom-configuredFully customizable cardsTaqMan Array CardsAllows the configuration of the TaqMan Array Cards with any predesigned assaysTaqMan Gene Expression Assays User Guide—TaqMan Array Cards5
1Chapter 1 Product informationOver of a TaqMan Array CardThis document provides guidance for preparing cDNA templates (see page 12) and protocols forperforming real-time PCR (qPCR) using a variety of compatible instruments and Master Mixes (seepage 13 and page 36).For more information about TaqMan Gene Expression Assays, see Appendix C, “Supplementalinformation”.Over of a TaqMan Array CardA TaqMan Array Card is a 384‑well microfluidic card that is prepared with dried-down TaqMan Assays. With an array card, gene expression is measured using the comparative Ct (ΔΔCt) method ofrelative quantitation. For more information about relative quantitation, see the Resources section atthermofisher.com/taqman.Advantages of a TaqMan Array Card include the following: Small-volume design that minimizes sample and reagent consumption. Streamlined reaction setup that saves time and reduces labor-intensive steps. Access to high-throughput, 384-well format without liquid-handling robotics. Two-fold discrimination detection at the 99.7% confidence level. Standardization across multiple samples in multiple laboratories.Each card can run 1 to 8 samples against 12 to 384 TaqMan Assay targets (including controls).2112343564781 Fill reservoir—Each reservoir is loaded with a sample-specific PCR reaction mix; the associated reaction wells fillwith that sample (8 total reservoirs)2 Fill reservoir strip—Support strip for fill reservoirs; removed before running the card3 Reaction well—Each well contains dried-down assay (384 total reaction wells)4 Reaction well row—A set of reaction wells that fill with the same sample-specific PCR reaction mix (8 total rows,each row associated with a single fill reservoir)6TaqMan Gene Expression Assays User Guide—TaqMan Array Cards
Chapter 1 Product informationContents and storage1Contents and storageTaqMan Array Cards contain dried-down TaqMan Gene Expression Assays according to theconfigured card layout.Table 1 Available card layoutsCat. No.Number of assays controlsNumber of samplesTaqMan Array Card 12434224711 18quadruplicatesTaqMan Array Card 16434679815 18triplicatesTaqMan Array Card 24434224923 18duplicatesTaqMan Array Card 32434679931 14triplicatesTaqMan Array Card 48434225347 18no replicatesFormatTaqMan Array Card 64434680063 12triplicatesTaqMan Array Card 96a434225995 14no replicatesTaqMan Array Card 96b434226195 12duplicatesTaqMan Array Card 1924346802191 11duplicatesTaqMan Array Card 3844342265380 41no replicates[1][2]Storage[1]2–8 C[2]See packaging for expiration date.Shipped at ambient temperature. See thermofisher.com/ambientshippping.Go to thermofisher.com/taqmanfiles to download the following files: Card layout files (HTML and CSV)—Show the position of each assay on the card. The HTML andCSV files contain identical information. Setup files (SDS in TXT format)—Contain the layout of the targets on the card. The files areimported into the software specific to your instrument to perform real-time PCR. Assay Information File (AIF in TXT format)—Describes the TaqMan Gene Expression Assays.See Understanding Your Shipment (Pub. No. MAN0017153) for detailed information about theAssay Information File.Note: During the download, you may be asked to enter specific order numbers or product information.TaqMan Gene Expression Assays User Guide—TaqMan Array Cards7
1Chapter 1 Product informationOrder TaqMan Array CardsThe run properties and the thermal protocol are not defined in the setup files and must be set up in theinstrument or software.Order TaqMan Array Cards Order fixed- or flexible-content TaqMan Array Cards.a. Go to thermofisher.com/taqmanarrays.b. Search by disease, pathway, biological process, or gene symbol. Order custom-configured TaqMan Array Cards.a. Go to thermofisher.com/customarraycards.b. Select a format, then use the Custom Array Configurator.Required materials and equipment not suppliedUnless otherwise indicated, all materials are available through thermofisher.com. "MLS" indicatesthat the material is available from fisherscientific.com or another major laboratory supplier. Catalognumbers that appear as links open the web pages for those products.Table 2Recommended products for isolation of RNAItemSourcethermofisher.com/rnaisolationKits for RNA isolationTable 3Recommended products for preparation of cDNAItemSourcecDNA kit or cDNA Master Mix, one of the following:8SuperScript IV VILO Master Mix11756050SuperScript IV VILO Master Mix with ezDNase Enzyme11766050High-Capacity cDNA Reverse Transcription Kit4368813TaqMan Gene Expression Assays User Guide—TaqMan Array Cards
Chapter 1 Product informationRequired materials and equipment not supplied1Table 4 PCR Master MixesItemSource(Recommended) TaqMan Fast Advanced Master Mix4444556TaqMan Gene Expression Master Mix4369016TaqMan Universal Master Mix II, with UNG4440038TaqMan Universal Master Mix II, no UNG4440047Table 5 Other materials and equipment required for the workflowItemSourceReal-time PCR instrument, one of the following:The instrument must be configured with the TaqMan Array Card block and heated cover.QuantStudio 7 Pro Real-Time PCR SystemQuantStudio 7 Flex Real-Time PCR SystemQuantStudio 12K Flex Real–Time PCR SystemContact your local salesofficeViiA 7 Real-Time PCR System7900HT Fast Real‑Time PCR SystemSoftwareQuantStudio Design and Analysis Software v2For use with the QuantStudio 7 Pro Real-Time PCR System.Can be used to analyze data files from all of the other compatible instruments.(Optional) Relative Quantification application (Optional) ExpressionSuite Softwarethermofisher.com/qpcrsoftwareAvailableat thermofisher.com/connectAvailable atthermofisher.com/expressionsuiteEquipmentThermal cycler, one of the following (or equivalent): Veriti Thermal Cycler SimpliAmp Thermal CyclerContact your local salesoffice ProFlex PCR SystemTaqMan Gene Expression Assays User Guide—TaqMan Array Cards9
1Chapter 1 Product informationRequired materials and equipment not suppliedItemSourceCentrifuge with custom buckets and card holders, one of the following: Sorvall centrifuge Megafuge centrifugeContact your local salesoffice Multifuge centrifugeSee the Resources section at thermofisher.com/taqmanarrays for a list ofcompatible centrifuges, rotors, and buckets.TaqMan Array Card Sealer(Referred to as Stylus Staker in some documents)Blank balance TaqMan Array Cards(Included with the instrument block upgrade / installation kit)Contact your local salesofficeContact your local salesofficeMicrocentrifugeMLSVortex mixerMLSFisher-Scientific21-379-00(Optional) Eppendorf MixMate (shaker)PipettesMLSMicropipettesMLSTubes, plates, and other consumablesthermofisher.com/plasticsPlastics consumablesAerosol-resistant barrier pipette tipsMLSDisposable glovesMLSReagentsNuclease-free waterRNase InhibitorN8080119RNaseOUT Recombinant Ribonuclease Inhibitor10777019TURBO DNA-free KitDNase I, Amplification Grade10MLSAM190718068015TaqMan Gene Expression Assays User Guide—TaqMan Array Cards
Chapter 1 Product informationWorkflow1WorkflowStart with cDNA templates from RNA samples (page 12) Combine cDNA and Master Mix (page 14) Prepare a TaqMan Array Card (page 15)(see page 25 for detailed procedures) Load the PCR reaction mix (page 27) Centrifuge the card (page 27) Seal the card (page 29) Set up and run the real-time PCR instrument (page 15) Analyze the results (page 16)TaqMan Gene Expression Assays User Guide—TaqMan Array Cards11
2Guidelines for preparation of cDNAGuidelines for isolation of high–quality RNA See Table 2 on page 8 for recommended RNA isolation kits. (Optional) Use DNase to ensure minimal genomic DNA contamination of the RNA.Guidelines for preparing cDNA templates See Table 3 on page 8 for recommended cDNA synthesis kits. Use the same reverse transcription procedure for all samples. For optimal reverse transcription, input RNA should be:– Free of inhibitors of reverse transcription (RT) and PCR– Dissolved in PCR-compatible buffer– Free of RNase activityNote: We recommend using RNase Inhibitor (Cat. No. N8080119) or RNaseOUT Recombinant Ribonuclease Inhibitor (Cat. No. 10777019).– Nondegraded total RNA (not applicable for double-stranded templates)IMPORTANT! Degradation of the RNA may reduce the yield of cDNA for some gene targets. For the input RNA amount, follow the recommendations provided by the cDNA kit. Small amounts of cDNA can be pre-amplified.Use TaqMan PreAmp Master Mix (Cat. No. 4391128) or TaqMan PreAmp Master Mix Kit (Cat.No. 4384267). Calculate the number of required reactions. Scale reaction components based on the singlereaction volumes, then include 10% overage, unless otherwise indicated. If using strip tubes to prepare cDNA templates, change to a new cap after each step or incubation. See your instrument user guide for detailed instructions about using plates, tubes, or strip tubes toprepare cDNA templates.12TaqMan Gene Expression Assays User Guide—TaqMan Array Cards
3Perform real-time PCR Procedural guidelines for performing real-time PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13Combine cDNA and Master Mix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14Prepare a TaqMan Array Card . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15Set up and run the real-time PCR instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15Analyze the results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16Procedural guidelines for performing real-time PCR Follow best-practices when preparing or performing PCR (see page 38). Prepare the real‑time PCR reactions in an area free of artificial templates and siRNA transfections.High‑copy‑number templates can easily contaminate the real‑time PCR reactions. Before preparing a TaqMan Array Card, review Appendix B, “Detailed procedures for preparationof a TaqMan Array Card”. Keep the card protected from light and stored as indicated until ready for use. Excessive exposureto light may affect the fluorescent probes of the dried-down assays in the card. Configure run documents according to the instructions provided in the real-time PCR instrumentresource documents. Load each fill reservoir with 100 µL of sample-specific PCR reaction mix.– Each fill reservoir contains a single sample as determined by the card layout.– The 100–µL volume ensures adequate filling of each reaction well. Volumes smaller than100 µL result in insufficiently filled cards. Equilibrate the card that is loaded with PCR reaction mix to room temperature before loading intothe real-time PCR instrument. Run the card within 72 hours of sealing the card if the following Master Mixes are used:– TaqMan Fast Advanced Master Mix– TaqMan Gene Expression Master Mix Run the card within 24 hours of sealing the card if the following Master Mixes are used:– TaqMan Universal Master Mix II, with UNG– TaqMan Universal Master Mix II, no UNG If the card is not run immediately, protect it from light and store at 2-8 C.TaqMan Gene Expression Assays User Guide—TaqMan Array Cards13
3Chapter 3 Perform real-time PCRCombine cDNA and Master MixGuidelines for quantity of cDNAUse the same quantity of cDNA sample for all reactions. Use a cDNA quantity of 30–1,000 ng per fillreservoir (0.3–10 ng/µL).The recommended quantity of cDNA depends on the expression level of the target genes and thenumber of target copies per well.Gene expression levelQuantity of cDNA per fill reservoir1,000 ng (10 ng/µL)[1]Low expressionModerate expression100–200 ng (1–2 ng/µL)Moderate to high expression30–50 ng (0.3–0.5 ng/µL)[1]The cDNA concentration will be high, so ensure that the cDNA is high-quality and without inhibitors.Combine cDNA and Master MixNote: Each fill reservoir (1 through 8) of the TaqMan Array Card is loaded with a sample‑specific PCRreaction mix according to the card layout.Thaw the cDNA samples on ice. Resuspend the cDNA samples by inverting the tube, then gentlyvortexing.1. Mix the Master Mix thoroughly but gently.Do not create bubbles in the Master Mix.2. Combine the cDNA and Master Mix in an appropriately-sized microcentrifuge tube according to thefollowing table:ComponentVolume per fill reservoir [1]cDNA sample nuclease‑free water55 µLMaster Mix (2X)55 µLTotal volume110 µL[1]Includes 10% overage.3. Vortex the tube to mix the contents thoroughly, then centrifuge briefly to collect the contents at thebottom of the tube.14TaqMan Gene Expression Assays User Guide—TaqMan Array Cards
Chapter 3 Perform real-time PCRPrepare a TaqMan Array Card3Prepare a TaqMan Array CardIMPORTANT! Before preparing a TaqMan Array Card, review Appendix B, “Detailed procedures forpreparation of a TaqMan Array Card”.1. Load each fill reservoir of the card with 100 μL of prepared PCR reaction mix.2. Centrifuge, then seal the filled card.Set up and run the real-time PCR instrumentSee the appropriate instrument user guide for detailed instructions to program the thermal-cyclingconditions or to run the card.Note: The instrument must be configured with a block appropriate for a card.1. Import the setup file (SDS in TXT format) into the real-time PCR instrument or software.See page 35 for instructions on how to import setup files.Note: The setup files are instrument–specific. See page 35 for more information.2. Select the cycling mode appropriate for the Master Mix.IMPORTANT! The cycling mode depends on the Master Mix that is used in the reaction.3. Set up the thermal protocol for your instrument.See “Thermal protocols” on page 36 for the thermal protocols for other Master Mixes.TaqMan Gene Expression Assays User Guide—TaqMan Array Cards15
3Chapter 3 Perform real-time PCRAnalyze the resultsNote: Your thermal protocols might differ from the following tables in this user guide.Table 6 TaqMan Fast Advanced Master Mix (ViiA 7 and compatible QuantStudio systemswith fast cycling mode)StepTemperatureTimeCyclesUNG incubation[1]50 C2 minutes1Enzyme activation[2]92 C10 minutes[3]1Denature95 C1 secondAnneal / Extend60 C20 seconds[1][2][3]40Optional, for optimal UNG activity.To activate AmpliTaq Fast DNA Polymerase.To completely dissolve the primers and probes on the card.Table 7 TaqMan Fast Advanced Master Mix (7900HT Fast Real-Time PCR Instrument with fastcycling mode)StepTemperatureTimeCyclesUNG incubation[1]50 C2 minutes1Enzyme activation[2]92 C10 minutes[3]1Denature97 C1 secondAnneal / Extend60 C20 seconds[1][2][3]40Optional, for optimal UNG activity.To activate AmpliTaq Fast DNA Polymerase.To completely dissolve the primers and probes on the card.4. Confirm that the reaction volume is set to 1 µL.5. Load the card into the real-time PCR instrument.6. Start the run.Analyze the resultsFor detailed information about data analysis, see the appropriate documentation for your instrument.Use the relative quantification (ΔΔCt) method to analyze results.A cutoff of 32 is recommended. If pre-amplification is used, the cutoff can be set to 29 or 30 to reducethe number of false positives.The general guidelines for analysis include: View the amplification plot; then, if needed:– Adjust the baseline and threshold values (if using the baseline threshold method of analysis).– Remove outliers from the analysis. In the well table or results table, view the Ct values for each well and for each replicate group.16TaqMan Gene Expression Assays User Guide—TaqMan Array Cards
Chapter 3 Perform real-time PCRAnalyze the results3Perform additional data analysis using any of the following software:SoftwareResourceRelative Quantification applicationthermofisher.com/connectExpressionSuite Software[1]thermofisher.com/expressionsuite[1]Can automatically define the baseline.For more information about real-time PCR, see page 43 or go to thermofisher.com/qpcreducation.Ct (Cq) values can be generated using the relative threshold algorithm (Crt).Use the relative threshold algorithm in your software. The relative threshold algorithm is available on thefollowing instruments: QuantStudio Real‑Time PCR Instruments ViiA 7 instrumentThe Relative Quantification application is also available on Thermo Fisher Connect.The Ct algorithm is recommended on the 7900HT Fast Real-Time PCR Instrument.See Introduction to Gene Expression Getting Started Guide (Pub. No. 4454239) for information about Ctand Crt.TaqMan Gene Expression Assays User Guide—TaqMan Array Cards17
3Chapter 3 Perform real-time PCRAnalyze the resultsAlgorithms for data analysisTable 8 Algorithm recommendations for TaqMan Array CardsAlgorithmRecommendationRecommended for the following instruments: QuantStudio Real‑Time PCR InstrumentsRelative threshold (Crt) ViiA 7 instrumentCan correct a variable baseline, which might be due to dried-downassays on the card being reconstituted at different rates.Threshold (Ct)Optional if used for analysis of established protocols.Recommended for 7900HT Fast Real-Time PCR Instrument.The relative threshold algorithm is available in the Relative Quantification application on Thermo Fisher Connect (thermofisher.com/connect).18TaqMan Gene Expression Assays User Guide—TaqMan Array Cards
A TroubleshootingTroubleshooting: After removing the card from packaging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19Troubleshooting: After loading PCR reaction mix into the card . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19Troubleshooting: After centrifuging the card . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20Troubleshooting: After running the card and reviewing run results . . . . . . . . . . . . . . . . . . . . . . . . . . . 20Troubleshooting: After removing the card from packagingObservationWater condenses on thereaction wells (optical side of thecard)Possible causeThe card was not at roomtemperature before beingremoved from the packaging.Recommended actionRemove condensation on the reaction wells bylightly blowing room temperature pressurizednitrogen or an air blower on the wells.IMPORTANT! Ensure that all watercondensation is rem
For Research Use Only. Not for use in diagnostic procedures. TaqMan Gene Expression Assays— TaqMan Array Cards USER GUIDE Publication Number 4400263 Revision G
