High‐Throughput Real‐Time Quantitative Reverse Transcription PCR

Angie L. Bookout1, Carolyn L. Cummins1, David J. Mangelsdorf1, Jean M. Pesola2, Martha F. Kramer2

1 Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, Texas, 2 Harvard Medical School, Boston, Massachusetts (preparation of RNA standards)
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 15.8
DOI:  10.1002/0471142727.mb1508s73
Online Posting Date:  February, 2006
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library


Extensive detail on the application of the real‐time quantitative polymerase chain reaction (QPCR) for the analysis of gene expression is provided in this unit. The protocols are designed for high‐throughput, 384‐well‐format instruments, such as the Applied Biosystems 7900HT, but may be modified to suit any real‐time PCR instrument. QPCR primer and probe design and validation are discussed, and three relative quantitation methods are described: the standard curve method, the efficiency‐corrected ΔCt method, and the comparative cycle time, or ΔΔCt method. In addition, a method is provided for absolute quantification of RNA in unknown samples. RNA standards are subjected to RT‐PCR in the same manner as the experimental samples, thus accounting for the reaction efficiencies of both procedures. This protocol describes the production and quantitation of synthetic RNA molecules for real‐time and non‐real‐time RT‐PCR applications.

Keywords: QPCR; quantitative PCR; real‐time PCR; reverse transcription PCR; RT‐PCR; RNA expression analysis

PDF or HTML at Wiley Online Library

Table of Contents

  • Strategic Planning
  • Basic Protocol 1: Standard Curve Method for Relative Quantification
  • Basic Protocol 2: Efficiency‐Corrected ΔCt Method
  • Alternate Protocol 1: Comparative or ΔΔCt Method
  • Support Protocol 1: Generation of RNA Standards for Absolute Quantification by Reverse Transcription PCR
  • Support Protocol 2: Design and Validation of SYBR Green and TaqMan Primer/Probe Sets
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
PDF or HTML at Wiley Online Library


Basic Protocol 1: Standard Curve Method for Relative Quantification

  • 20 ng/µl experimental cDNA samples (concentration based on RNA input for cDNA synthesis; see unit 15.5)
  • Dilution series of standard cDNAs (see recipe) or dilution series of [35S]RNA standards (see protocol 4)
  • No‐template control sample (NTC; prepared at the same time as the cDNA samples using molecular‐biology‐grade water instead of RNA; see )
  • No‐reverse‐transcriptase control samples (–RT; prepared at the same time as the cDNA samples using molecular‐biology‐grade water instead of reverse transcriptase; see )
  • 2× SYBR Green or TaqMan mix containing ROX (Applied Biosystems, Bio‐Rad, Invitrogen, Sigma, or see recipe for 2× SYBR Green mix)
  • Primer mixes, 1.25 µM each forward and reverse primer (see recipe and protocol 5), for each reference gene and GOI to be tested
  • 5 µM TaqMan probe (for TaqMan protocol only; see recipe and protocol 5)
  • Molecular‐biology‐grade water (nucleic acid and nuclease free)
  • 8‐tube PCR tube strips (optional, but recommended; can be of low quality since they will only be used for mixing reaction components; ISC Bioexpress)
  • 96‐well PCR tube racks (optional, but recommended; ISC Bioexpress)
  • Digital multichannel pipettor, 8‐ or 12‐channel, 5‐ to 100‐µl capacity (recommended)
  • Centrifuge with swinging‐bucket rotor and microtiter plate carriers
  • 384‐well optical reaction plates (Applied Biosystems)
  • Optical adhesive covers (Applied Biosystems)
  • Real‐time thermal cycler: e.g., Applied Biosystems 7900HT
  • Microsoft Excel or spreadsheet program with equivalent statistical features

Basic Protocol 2: Efficiency‐Corrected ΔCt Method

  • cDNA or DNA fragment containing target sequence
  • Vector containing T7, T3, or SP6 RNA polymerase promoter
  • Appropriate restriction enzyme (unit 3.1) for linearizing plasmid
  • 25:24:1 (v/v/v) phenol/chloroform/isoamyl alcohol
  • 49:1 (v/v) chloroform/isoamyl alcohol
  • 3 M sodium acetate ( appendix 22)
  • 100% ethanol
  • Nuclease‐free water
  • 0.5 µg/ml sheared salmon sperm DNA
  • 1 M Tris·Cl, pH 7.5 ( appendix 22)
  • 1 M MgCl 2 ( appendix 22)
  • 1 M DTT ( appendix 22)
  • 500 µM 4NTP mix: 500 µM each ATP, CTP, GTP, and UTP
  • 600 Ci/mmol (10 mCi/ml) [α‐35S]CTP or UTP
  • Bovine serum albumin (BSA)
  • Spermidine (for SP6 only)
  • T7, T3, or SP6 RNA polymerase (unit 3.8)
  • 1 U/µl RNase‐free DNase I (e.g., Promega)
  • RNeasy Mini Kit (Qiagen) or equivalent
  • 10% trichloroacetic acid (TCA; see recipe), ice cold
  • 100% methanol, ice cold
  • Universal scintillation cocktail (preferably biodegradable, e.g., Ecoscint A, National Diagnostics)
  • 1.5‐ml screw‐cap microcentrifuge tubes
  • DE81 paper (Whatman)
  • Filter paper
  • Glass fiber filters (Whatman GF/C 24‐mm discs)
  • Transparent plastic wrap
  • Forceps
  • Heat lamp (optional)
  • 250‐ml glass or metal beaker
  • Liquid scintillation counter and vials
  • Additional reagents and equipment for subcloning (unit 3.16), plasmid minipreps (unit 1.6), digestion with restriction endonucleases (unit 3.1), agarose gel electrophoresis (unit 2.5), purification of DNA (unit 2.1), phage RNA polymerase reactions (units 3.4& 3.8), agarose‐formaldehyde or glyoxal gel electrophoresis (unit 4.9), and drying and imaging of gels ( 3.NaN)
CAUTION: To minimize the risk of radioactive contamination, use screw‐cap microcentrifuge tubes with cap gaskets and filtered pipet tips for all manipulations of radioactive solutions. Wear gloves and dispose of all 35S‐contaminated material properly. See appendix 1F for more details on handling radioactivity.CAUTION: Phenol, chloroform, and trichloroacetic acid are hazardous (see appendix 1H).NOTE: For all procedures involving RNA, use reagents and solutions that are free of contaminating RNases, DNases, and nucleic acids, and follow other guidelines for handling RNA (unit 4.1).

Alternate Protocol 1: Comparative or ΔΔCt Method

  • Primer/probe design software (Primer Express, Applied Biosystems)
PDF or HTML at Wiley Online Library



Literature Cited

   Alberts, B., Bray, D., Lewis, J., Raff, M., Roberts, K., and Watson, J.D. 1994. The cell nucleus. In Molecular Biology of the Cell, 3rd ed., p. 370. Garland Publishing, New York.
   Applied Biosytems. 2001a. ABI Prism 7900HT User Manual, rev. 4. Applied Biosystems, Foster City, Calif.
   Applied Biosystems. 2001b. TaqMan human endogenous control plate: Protocol: Revision C. http://docs.appliedbiosystems.com/pebiodocs/04308134.pdf. Applied Biosystems, Foster City, Calif.
   Applied Biosystems. 2002a. Real‐time PCR vs. traditional PCR. https://www.appliedbiosystems.com/support/tutorials/pdf/rtpcr_vs_tradpcr.pdf. Applied Biosystems, Foster City, Calif.
   Applied Biosystems. 2002b. Designing TaqMan MGB probe and primer sets for gene expression using Primer Express software. http://www.appliedbiosystems.com/support/tutorials/pdf/taqman_mgb_primersprobes_for_gene_expression.pdf. Applied Biosystems, Foster City, Calif.
   Applied Biosystems. 2003. Creating standard curves with genomic DNA or plasmid DNA templates for use in quantitative PCR. https://www.appliedbiosystems.com/support/tutorials/pdf/quant_pcr.pdf. Applied Biosystems, Foster City, Calif.
   Brown, T.A. 2002. How genomes function. In Genomes, 2nd ed. (S. Carlson, ed.) section 10.4. John Wiley & Sons, Hoboken, N.J.
   Clontech. 2002. Control RNA for microarray experiments. Clontechniques XVII:6. http://www.clontech.com/clontech/archive/APR02UPD/pdf/ControlRNA.pdf. Clontech, Palo Alto, Calif.
   Dheda, K., Huggett, J.F., Bustin, S.A., Johnson, M.A., Rook, G., and Zumla, A. 2004. Validation of housekeeping genes for normalizing RNA expression in real‐time PCR. BioTechniques 37:112‐119.
   Guo, D., Henriksson, R., and Hedman, H. 2001. The iCycler iQ detection system for evaluating reference gene expression in normal human tissue, rev. A. Amplification 2804. http://www.bio‐rad.com/LifeScience/pdf/Bulletin_2804.pdf. Bio‐Rad, Hercules, Calif.
   Iyer, V. and Struhl, K. 1996. Absolute mRNA levels and transcriptional initiation rates in Saccharomyces cerevisiae. Proc. Nat. Acad. Sci. U.S.A. 93:5208‐5212.
   Kramer, M.F. and Coen, D.M. 1995. Quantification of transcripts from the ICP4 and thymidine kinase genes in mouse ganglia latently infected with herpes simplex virus. J. Virol. 69:1389‐1399.
   Morrison, T.B., Weis, J.J., and Wittwer, C.T. 1998. Quantification of low‐copy transcripts by continuous SYBR Green I monitoring during amplification. BioTechniques 24:954‐962.
   Mullis, K.B. and Faloona, F.A. 1987. Specific synthesis of DNA in vitro via a polymerase‐catalyzed chain reaction. Methods Enzymol. 155:335‐350.
   Novoradovskaya, N., Payette, T., Novoradovsky, A., Braman, J., Chin, N., Pergamenschikov, A., Fero, M., and Botstein, D. 2000. Pooled, high‐quality reference RNA for human microarrays. Strategies 13:121‐122. http://www.stratagene.com/news/newsletter.aspx?iid=6. Strategene, La Jolla, Calif.
   Pfaffl, M.W. 2004. Quantification strategies in real‐time PCR. In IUL Biotechnology Series, No. 5: A‐Z of Quantitative PCR (S.A. Bustin, ed.) pp. 87‐120. International University Line, La Jolla, Calif.
   Roche Applied Science. 2002. Selection of housekeeping genes. Technical Note No. LC 15/2002. http://www.roche‐applied‐science.com/lightcycler‐online/lc_support/pdfs/lc_15.pdf. Roche Applied Science, Indianapolis, Ind.
   Schenborn, E.T. and Mierendorf, R.C. Jr. 1985. A novel transcription property of SP6 and T7 RNA polymerases: Dependence on template structure. Nucl. Acid. Res. 13:6223‐6236.
   Schmittgen, T.D. and Zakrajsek, B.A. 2000. Effect of experimental treatment on housekeeping gene expression: Validation by real‐time, quantitative RT‐PCR. J. Biochem. Biophys. Methods 46:69‐81.
   Shoemaker, J.P., Garland, C.W., and Steinfeld, J.I. 1974. Experiments in Physical Chemistry, pp. 34‐39. McGraw‐Hill, New York.
   Suzuki, T., Higgins, P.J., and Crawford, D.R. 2000. Control selection for RNA quantitation. BioTechniques 29:332‐337.
   Tyagi, S. and Kramer, F.R. 1996. Molecular beacons: Probes that fluoresce upon hybridization. Nat. Biotechnol. 14:303‐308.
   Vandesompele, J., De Preter, K., Pattyn, F., Poppe, B., Van Roy, N., De Psepe, A., and Speleman, F. 2002. Accurate normalization of real‐time quantitative RT‐PCR data by geometric averaging of multiple internal control genes. Genome Biol. 3:1‐12.
   Whitcombe, D., Theaker, J., Guy, S.P., Brown, T., and Little, S. 1999. Detection of PCR products using self‐probing amplicons and fluorescence. Nat. Biotechnol. 17:804‐807.
   Whittwer, C.T., Herrmann, M.G., Moss, A.A., and Rasmussen, R.P. 1997. Continuous fluorescence monitoring of rapid cycle DNA amplification. BioTechniques 22:130‐138.
Key References
   Ambion. 2001. The top 10 most common quantitative RT‐PCR pitfalls. Technotes Newsletter 8:8. Ambion, Houston, Tex.
  A short, but useful checklist of critical considerations for performing any type of reverse transcription PCR.
   Applied Biosystems. 1997. Relative quantitation of gene expression: ABI PRISM 7700 Sequence Detection System: User Bulletin #2: Rev B. Applied Biosystems, Foster City, Calif.
  This bulletin outlines both the standard curve and ΔΔCt methods and shows, by comparing data obtained using both calculations, that the resulting values are very similar regardless of the assay.
   Applied Biosytems. 2001a. See above.
  This instrument manual contains explanations about the transformation of fluorescence signal into Ct data in addition to outlining the proper method of baseline and threshold settings for ABI machines. Users should consult their specific instrument manuals, since each type of instrumentation will require knowledge of slightly different terminology and parameters.
   Livak, K.J. and Schmittgen, T.D. 2001. Analysis of relative gene expression data using real‐time quantitative PCR and the 2−ΔΔCt method. Methods 25:402‐408.
  This article presents a detailed review of the derivation of the mathematical applications described in this unit.
Internet Resources
  NCBI Web site.
  Ensembl Web site.
  The Gene Quantification Web site contains a host of information concerning QPCR.
  The Primer Bank database, hosted by Harvard University, contains user‐submitted primer sequences for several mouse and human genes.
  The Quantitative PCR Primer Database (QPPD), maintained by the National Cancer Institute, contains primer and probe sequences for mouse and human genes collected from articles cited in PubMed.
  Contains numerous, detailed articles and technical bulletins regarding transcription and general RNA handling issues.
  Contains technical manuals with detailed protocol tips and troubleshooting for transcription applications
PDF or HTML at Wiley Online Library