TaqPath 1-Step RT-qPCR Master Mix, CG - Thermo Fisher Scientific
USER GUIDETaqPath 1-Step RT-qPCR Master Mix, CGCatalog Numbers A15299, A15300Publication Number MAN0007959Revision 1.0For Laboratory Use.
For Laboratory Use.The information in this guide is subject to change without notice.Manufacturing Site:Life Technologies Corporation 7335 Executive Way Frederick, MD 21704 USADISCLAIMERLIFE TECHNOLOGIES CORPORATION AND/OR ITS AFFILIATE(S) DISCLAIM ALL WARRANTIES WITH RESPECT TO THIS DOCUMENT, EXPRESSED OR IMPLIED,INCLUDING BUT NOT LIMITED TO THOSE OF MERCHANTABILITY, FITNESS FOR A PARTICULAR PURPOSE, OR NON-INFRINGEMENT. TO THE EXTENTALLOWED BY LAW, IN NO EVENT SHALL LIFE TECHNOLOGIES AND/OR ITS AFFILIATE(S) BE LIABLE, WHETHER IN CONTRACT, TORT, WARRANTY, ORUNDER ANY STATUTE OR ON ANY OTHER BASIS FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE OR CONSEQUENTIAL DAMAGES INCONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING BUT NOT LIMITED TO THE USE THEREOF.LIMITED USE LABEL LICENSE No. 475: General Purpose ReagentsNOTICE TO PURCHASER: The purchase of this product conveys to the purchaser the limited, non-transferable right to use the purchased amount of theproduct only to perform internal research and development for the sole benefit of the purchaser. No right to transfer or resell this product or any of itscomponents is conveyed expressly, by implication, or by estoppel. This product is for internal research and development purposes only and is not for use incommercial applications of any kind, including, without limitation, quality control and commercial services such as reporting the results of purchaser'sactivities for a fee or other form of consideration. For obtaining additional rights, please contact outlicensing@lifetech.com or Out Licensing, Life Technologies,5791 Van Allen Way, Carlsbad, California 92008.LIMITED USE LABEL LICENSE No. 469: PCR (Roche Intellectual Property) General Purpose ReagentNOTICE TO PURCHASER: For research purposes only. Diagnostic uses require a separate license from Roche.TRADEMARKSThe trademarks mentioned herein are the property of Life Technologies Corporation and/or its affiliate(s) or their respective owners. Adobe and Reader areregistered trademarks of Adobe Systems Incorporated. 2013 Life Technologies Corporation. All rights reserved.
ContentsAbout This Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7Revision history . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7Purpose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 CHAPTER 1Product Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9Product description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9Purpose of the product . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9Starting template . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9About this protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9About the kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10Storage and stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10Kit components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10General laboratory supplies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10Real-Time PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11Real-time PCR Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11Thermal-cycling conditions for your real-time PCR system . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 CHAPTER 2Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13For more information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13Before you begin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13Assays and thermal-cycling conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13Prevent contamination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13User-supplied materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13Workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14Prepare samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15Storage conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15Design the experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15User-defined assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15Select your experiment type . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15Starting template . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15RT-qPCR guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16Determine the number of reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17Set up a plate document or experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18For fast real-time PCR systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18For standard real-time PCR systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18TaqPath 1-Step RT-qPCR Master Mix3
ContentsPrepare RT-qPCR reaction mix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19Fast real-time PCR systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19Standard real-time PCR systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20Run the RT-qPCR plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21Analyze the data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21About baseline and threshold values . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21For more information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 APPENDIX AAssay Design Guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . 23Workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23Amplicon site selection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24General amplicon site selection guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24If the gene does not contain introns . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24Probe and primer design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25General probe design guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25General primer design guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25Calculation of oligonucleotide concentrations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26Calculate oligonucleotide concentrations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26An example calculation of primer concentration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27An example calculation of probe concentration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27Determine optimal primer concentrations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Purpose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Primer concentrations to test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Prepare and run the RT-qPCRs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .28282828Determine optimal probe concentration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Purpose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Probe concentrations to test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Prepare and run the RT-qPCRs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .29292929Evaluate the results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 APPENDIX BPCR Good Laboratory Practices . . . . . . . . . . . . . . . . . . . 31Sample preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31Preventing contamination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .False positives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Uracil-N glycosylase (UNG) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Prevention of PCR product carryover . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Fluorescent contaminants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .43232323333TaqPath 1-Step RT-qPCR Master Mix
Contents APPENDIX CSafety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35Chemical safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35Biological hazard safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36Documentation and Support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37Related documentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37Obtaining SDSs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38Obtaining Certificates of Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38Obtaining support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38Limited product warranty . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38Medical Device Symbols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39Glossary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41TaqPath 1-Step RT-qPCR Master Mix5
Contents6TaqPath 1-Step RT-qPCR Master Mix
About This GuideIMPORTANT! Before using this product, read and understand the information in the“Safety” appendix in this document.Revision historyRevisionDateDescription1.010 September 2013User Guide to accompany new product introduction.PurposeThe TaqPath 1‐Step RT‐qPCR Master Mix User Guide provides a general protocol forperforming 1‐step real‐time reverse transcription quantitative PCR (RT‐qPCR) usingTaqPath 1‐Step RT‐qPCR Master Mix.TaqPath 1 Step RT-qPCR Master Mix7
About This GuidePurpose8TaqPath 1 Step RT-qPCR Master Mix
1Product InformationProduct descriptionPurpose of theproductUse the TaqPath 1‐Step RT‐qPCR Master Mix with any gene‐specific primer/probe setto amplify RNA or DNA target sequences. This master mix in combination with a userdefined and supplied assay allows the user to perform 1‐step RT‐qPCR for thefollowing types of experiments: Presence/absence – An endpoint experiment that indicates the presence orabsence of a specific nucleic acid sequence (target) in a sample. The actualquantity of target is not determined. Presence/absence experiments are commonlyused to detect the presence or absence of a pathogen, such as a viral or bacterialpathogen. (Presence/absence experiments are also referred to as plus/minusexperiments.) Standard curve – A type of quantification experiment that determines theabsolute target quantity in samples. With the standard curve method, the real‐time PCR system software measures amplification of the target in samples and ina standard dilution series. Data from the standard dilution series are used togenerate the standard curve. Using the standard curve, the software interpolatesthe absolute quantity of target in the samples. Standard curve experiments arecommonly used for quantifying viral load. (Standard curve experiments are alsoreferred to as absolute quantification or AQ experiments.)You can also perform a standard curve experiment without running standards, ifyou only want to collect the quantification (CT) values.Note: A quantification experiment is a real‐time experiment that measures thequantity of a target nucleic acid sequence (target) during each amplification cycleof the polymerase chain reaction (PCR).Starting templateYou can use this protocol for both RNA and DNA targets. During thermal cycling, thereverse transcription step will not affect performance with DNA targets.About this protocolTaqPath 1‐Step RT‐qPCR Master Mix has been optimized for use with primers andhydrolysis probes. This protocol describes how to perform gene expressionexperiments with the TaqPath 1‐Step RT‐qPCR Master Mix.Because analysis methods vary greatly between applications, this protocol providesgeneral guidelines for analyzing data generated from experiments that use TaqPath 1‐Step RT‐qPCR Master Mix in user defined assays. For more detailed informationabout data analysis or the procedures outlined in this protocol, refer to the appropriatedocumentation for your instrument.TaqPath 1-Step RT-qPCR Master Mix9
1Chapter 1 Product InformationAbout the kitAbout the kitStorage andstabilityStore the TaqPath 1‐Step RT‐qPCR Master Mix at –25 C to –15 C. The master mix willnot freeze at –25 C to –15 C; gelling may occur.Performance of the TaqPath 1‐Step RT‐qPCR Master Mix is guaranteed until theexpiration date printed on the package and bottle labels.Note: It is normal for this Master Mix to have a faint yellow hue.Kit componentsThe TaqPath 1‐Step RT‐qPCR Master Mix contains: Fast DNA Polymerase Thermostable MMLV enzyme Uracil‐N glycosylase (UNG) dNTPs including dUTP RNase inhibitor ROX dye (passive reference) Buffer components optimized for maximum sensitivity and tolerance to severalcommon RT‐qPCR inhibitorsThe TaqPath 1‐Step RT‐qPCR Master Mix is supplied at a 4X concentration and isavailable in the following quantities:CatalogNo.ProductTaqPath 1-Step RT-qPCR Master Mix, CGVolumeNo. of 20 µLreactionsA152995 1 mL1000A153001 10 mL2000General laboratory suppliesAll items are available from major laboratory suppliers (MLS).Item10SourceDisposable glovesMLSPipette tips (aerosol-resistant, nuclease-free)MLSPipettes (positive/air-displacement or multichannel)MLSLiquid reservoirs (RNase-free)MLSVortexerMLSCentrifugeMLSOptical reaction platesMLSOptical adhesive coversMLSRT-PCR grade waterMLSTaqPath 1-Step RT-qPCR Master Mix
Chapter 1 Product InformationReal-Time PCR1Real-Time PCRReal-time PCRSystemsYou must perform the PCR step on a real‐time quantitative PCR system. Traditionalthermal cyclers cannot be used because they cannot detect and record fluorescentsignal data generated by the cleavage of hydrolysis probes. Life TechnologiesCorporation offers the following real‐time quantitative PCR systems for use in assaydevelopment:Real-Time PCR System†QuantStudio Dx Real-Time PCR System7500 Fast Dx Real-Time PCR SystemAvailable run modesStandard/FastFast† For more information on available real-time PCR systems, accessories and consumables, ions for yourreal-time PCRsystemThe TaqPath 1‐Step RT‐qPCR Master Mix can be run in either Fast or Standardcycling systems, provided the thermal‐cycling profile and run mode are correctly setfor the instrument being used. See “Set up a plate document or experiment” onpage 18 for thermal cycling profiles for both fast and standard modes. Run mode – The run mode defines the ramp rate that is used to heat or cool thesample block between temperature changes. Thermal‐cycling profile – The thermal‐cycling profile defines the temperatureand time for each step. Be sure to use the appropriate thermal‐cycling profile foryour system.TaqPath 1-Step RT-qPCR Master Mix11
112Chapter 1 Product InformationReal-Time PCRTaqPath 1-Step RT-qPCR Master Mix
2OverviewFor moreinformationMethodsThis chapter provides a general protocol for performing 1‐Step RT‐qPCR usingTaqPath 1‐Step RT‐qPCR Master Mix. This protocol is suggested as a starting point.Optimal reaction conditions—incubation times and temperatures, primer/probeconcentration, and the amount of template—can vary and should be optimized. Before you begin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 User‐supplied materials. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 Workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 Design the experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 RT‐qPCR guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 Analyze the data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22For more detailed information about the procedures outlined in this protocol, refer tothe appropriate documentation for your instrument. See “Documentation andSupport” on page 37.Before you beginAssays andthermal-cyclingconditionsEach assay should be independently optimized and validated to ensure appropriateperformance. Validate your assays and re‐optimize your thermal‐cycling conditions asneeded.The reverse transcriptase enzyme contained in this kit is purified from E. coliexpressing a proprietary version of the MMLV pol gene (GenBank accession no. J02255)expressed from pET‐24( ) expression vector. It is possible that a minimal amount of theexpression vector could be carried over into the final master mix formulation. If youare targeting MMLV, a related virus, or any of the plasmid sequence, we recommenddesigning primer sequences not contained in the expression eview Appendix B, “PCR Good Laboratory Practices” on page 31. Nuclease‐free water and buffers User‐defined expression assay Reaction plates and accessories for the RT‐qPCR system employed General laboratory equipmentTaqPath 1 Step RT-qPCR Master Mix13
2Chapter 2 MethodsWorkflowWorkflowThe procedure for 1‐Step RT‐qPCR is outlined in the following workflow diagram.Prepare samples (page 15)Design the experiment (page 15)Select your experiment type (page 15)Determine the number of reactions (page 17)Set up a plate document or experimentFast system (page 18) Standard system (page 18)Prepare RT-qPCR reaction mixFast system (page 19) Standard system (page 20)Run the RT-qPCR plate (page 21)Analyze the data (page 21)14TaqPath 1 Step RT-qPCR Master Mix
Chapter 2 MethodsPrepare samples2Prepare samplesIsolate and purify the target nucleic acid samples according to your laboratorypractices. Life Technologies offers products appropriate for nucleic acid purificationfrom a spectrum of sample types.Storage conditionsStore the prepared samples at –85 C to –68 C in RT‐PCR grade water. If you diluteyour samples, use TE buffer or RT‐PCR grade water as the diluent.Design the experimentUser-definedassaysTo design your own assay for use with the TaqPath 1‐Step RT‐qPCR Master Mix, seeAppendix A, “Assay Design Guidelines” on page 23 for more information.Note: The term assay refers to the primer and probe set.Select yourexperiment typeSelect one of the following experiment types: Presence/absence Standard curveStarting templateThis protocol can be used for both RNA and DNA targets as the reverse transcriptionstep will not affect thermal cycling performance with DNA targets. However, werecommend using TaqPath qPCR Master Mix (Cat. no. A15297) when designingexperiments to assay DNA targets.TaqPath 1 Step RT-qPCR Master Mix15
2Chapter 2 MethodsDesign the experimentRT-qPCRguidelinesItemAssays (primer and probe set)GuidelineKeep all assays in the freezer, protected from light, until you are ready to use them.Excessive exposure to light may affect the fluorescent probes.Just before use, allow the assays to thaw on ice.At initial use, aliquot the assays to avoid multiple freeze/thaw cycles.TaqPath 1 Step RT-qPCRMaster MixKeep the master mix in the freezer, protected from light, until you are ready to use it.Just before use, allow the master mix to thaw on ice.Note: The master mix does not freeze at –25 C to –15 C but gelling may occur.Thawing the master mix on ice allows the master mix to return to its liquid state.Storing combined master mixand assayYou can combine the TaqPath 1 Step RT-qPCR Master Mix and the assay ahead of timeand store at –25 C to –15 C for short periods. Stability varies depending on the assay,but storage for up to 4 weeks of the combined master mix and assay has been observedto have minimal effect on performance.(For standard curveexperiments)Standards are critical for accurate analysis of run data. Mistakes or inaccuracies inmaking the dilutions directly affect the quality of the results. The quality of pipettes andtips and the care used in measuring and mixing dilutions affect accuracy. Use TE bufferor RT-qPCR Grade Water to prepare the standard dilution series.StandardsNo-RT controlIf you are concerned that your 1-step real-time RT-qPCR is detecting genomic DNArather than a particular RNA species, you can run a no-RT control reaction with theTaqPath 1 Step RT-qPCR Master Mix. To run a no-RT control: Heat-kill the RT enzymeby heating one aliquot of master mix at 95 C for 5 minutes before mixing it with theassay and sample. The PCR hot-start mechanism will reactivate after the master mixhas cooled to room temperature.Thermal-cycling temperaturerangesThe optimal temperatures for reverse transcription and annealing are recommended inthis protocol (see “Set up a plate document or experiment” on page 18). However, insome instances you may wish to alter the temperatures; testing has shown that the: RT enzyme will function best in the range of 48–55 C Annealing temperature should be in the range of 56–62 CNote: Be sure the annealing temperature is consistent with the meltingtemperature (Tm) of your primer designs. For guidelines on designing primers, see“Probe and primer design” on page 25.Multiplexing16TaqPath 1 Step RT-qPCR Master Mix is designed to accommodate running multipleassays simultaneously. For guidelines on designing multiplex reactions, refer to FactorsInfluencing Multiplex Real-Time PCR, “Related documentation” on page 37.TaqPath 1 Step RT-qPCR Master Mix
Chapter 2 MethodsDesign the experimentDetermine thenumber e the total number of reactions in your experiment. For each experiment type,you need the following types of reactions:Reaction typeUnknownDescriptionA well that contains: Sample (DNA or RNA in which the presence of a target is unknown) TaqPath 1 Step RT-qPCR Master Mix Assay of choiceStandardcurveExogenousInternal positivecontrol (IPC)A short synthetic DNA template that you can add to the reactions to distinguishbetween true negative results and reactions affected by PCR inhibitors, incorrectassay setup, or a reagent or instrument failure.No amplificationcontrol (NAC)A well that contains all reaction components except the unknown sample and IPC.Alternatively, the well may contain the IPC plus a blocking agent for the IPC. Noamplification should occur in NAC wells.No templatecontrol (NTC)A well that contains all PCR components except the unknown sample. Only theIPC should amplify in NTC wells.ReplicateA well that is identical to another; it contains identical components and volumes.Life Technologies recommends performing at least three replicates of eachreaction.UnknownA well that contains: Sample (DNA or RNA in which the quantity of the target is unknown) TaqPath 1 Step RT-qPCR Master Mix Assay of choiceStandardA well that contains DNA of a known standard quantity; used in quantificationexperiments to generate standard curves.Note: You can perform a standard curve experiment without running standards,if you only want to collect the CT values.Standard dilutionseriesA set of standards containing a range of known quantities. The standard dilutionseries is prepared by serially diluting standards.No templatecontrol (NTC)A negative control well that contains water or buffer instead of sample. Noamplification of the target should occur in negative control wells.ReplicateA well that is identical to another; it contains identical components and volumes.Life Technologies recommends performing at least three replicates of eachreaction.TaqPath 1 Step RT-qPCR Master Mix17
2Chapter 2 MethodsSet up a plate document or experimentSet up a plate document or experimentFor fast real-timePCR systemsIn the real‐time PCR system software, set up a plate document or experiment using thefollowing parameters: Sample volume: 20 μL Auto Increment Settings: Accept the default value Data Collection: Accept the default value Ramp Rate Settings: Accept the default value Run mode: Use the default run mode for your system and sample block module(that is, Fast mode on Fast instruments and standard mode on standardinstruments). Thermal‐cycling conditions for sample volumes 30 μL:StepUNG ion‡AmplificationStageNo. ofcyclesTemperature1125 C2 minutes150 C†15 minutes3195 C‡2 minutes44095 C3 seconds60 C30 seconds2Time† Reverse transcription works best between 48 C and 55 C.‡ Required for RT inactivation, initial denaturation, and to activate the DNA polymerase.For standard realtime PCR systemsIn the real‐time PCR system software, set up a plate document or experiment using thefollowing parameters: Sample volume: 50 μL Auto Increment Settings: Accept the default value Data Collection: Accept the default value Ramp Rate Settings: Accept the default value Run mode: Standard Thermal‐cycling conditions for sample volumes 30 μL:StepUNG ion‡AmplificationStageNo. ofcyclesTemperature1125 C2 minutes150 C†15 minutes3195 C‡2 minutes44095 C15 seconds60 C60 seconds2Time† Reverse
The TaqPath 1‐Step RT‐qPCR Master Mix User Guide provides a general protocol for performing 1‐step real‐time reverse transcription quantitative PCR (RT‐qPCR) using TaqPath 1‐Step RT‐qPCR Master Mix. Revision Date Description 1.0 10 September 2013 User Guide to accompany new product introduction.
Introduction to Quantitative PCR Whether you are a novice or experienced user, our goal is to ensure that you are running quantitative PCR (QPCR) experiments quickly, efficiently, and affordably. Our Mx family of QPCR Systems, MxPro QPCR Software, premiere QPCR Systems Service Program, complete line of QPCR and QRT-PCR reagents, and Fast
FDA-released Instructions for Use with removal of the licensing statement covering Limited Use Label Licenses on page 2. H.0 10 November 2020 The instructions for using the TaqPath COVID-19 Combo Kit Advanced were updated to indicate that the kit is now compatible with all instruments.
grade step 1 step 11 step 2 step 12 step 3 step 13 step 4 step 14 step 5 step 15 step 6 step 16 step 7 step 17 step 8 step 18 step 9 step 19 step 10 step 20 /muimn 17,635 18,737 19,840 20,942 22,014 22,926 23,808 24,689 325,57! 26,453 /2qsohrs steps 11-20 8.48 9.0! 9.54 10.07 10.60 11.02 11.45 11.87 12.29 12.72-
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2 Stratagene QPCR Human Reference Total RNA INTRODUCTION Stratagene QPCR Human Reference Total RNA is a high-quality control for quantitative PCR gene-expression analysis.
time quantitative PCR (qPCR). As miRNA analysis is a fast changing research field, we have introduced novel technological approaches and compared them to existing qPCR profiling methodologies. qPCR also remains the method of choice for validating results obtained from whole-genome screening (e.g. with microarray).
Human Factors and Usability Engineering – Guidance on the regulation of Medical Devices Including Drug-device Combination Products in Great Britain Version 2.0 January 2021 . Human Factors and Usability Engineering – Guidance for Medical Devices Including Drug-device Combination Products MHRA September 2017 v1.0 Page 2 of 35 Contents 1 Introduction and context . 4 2 The regulatory .
