Real‐Time PCR

Dean Fraga1, Tea Meulia2, Steven Fenster3

1 College of Wooster, Wooster, Ohio, 2 Ohio Agricultural Research and Development Center, Wooster, Ohio, 3 Fort Lewis College, Durango, Colorado
Publication Name:  Current Protocols Essential Laboratory Techniques
Unit Number:  Unit 10.3
DOI:  10.1002/9780470089941.et1003s08
Online Posting Date:  February, 2014
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Abstract

Real‐time PCR is a recent modification to the polymerase chain reaction that allows precise quantification of specific nucleic acids in a complex mixture by fluorescent detection of labeled PCR products. Detection can be accomplished using specific as well as nonspecific fluorescent probes. Real‐time PCR is often used in the quantification of gene expression levels. Prior to using real‐time PCR to quantify a target message, care must be taken to optimize the RNA isolation, primer design, and PCR reaction conditions so that accurate and reliable measurements can be made. This short overview of real‐time PCR discusses basic principles behind real‐time PCR, some optimization and experimental design considerations, and how to quantify the data generated using both relative and absolute quantification approaches. Useful Web sites and texts that expand upon topics discussed are also listed. Curr. Protoc. Essential Lab. Tech. 8:10.3.1‐10.3.40. © 2014 by John Wiley & Sons, Inc.

Keywords: quantifying gene expression; real‐time PCR (polymerase chain reaction); cDNA; PCR primer design; Taq polymerase; SYBR Green; PCR

     
 
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Table of Contents

  • Overview and Principles
  • Strategic Questions
  • Strategic Planning
  • Safety Considerations
  • Protocols
  • Basic Protocol 1: Synthesis of cDNA by Reverse Transcription
  • Basic Protocol 2: Real‐Time PCR Amplification and Analysis
  • Support Protocol 1: Determination of Amplification Efficiency
  • Support Protocol 2: Analyzing Results Using the Pfaffl Method to Calculate Fold Induction
  • Support Protocol 3: Serial Dilution for Standard Curve
  • Understanding Results
  • Troubleshooting
  • Variations
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Synthesis of cDNA by Reverse Transcription

  Materials
  • Purified total RNA dissolved in DEPC‐treated water
  • First strand cDNA synthesis primers (50 μM)
  • 10 mM dNTP (free nucleotides) mix (unit 3.3)
  • DEPC‐treated H 2O (unit 8.2)
  • 10× RT buffer
  • 25 mM MgCl 2
  • 0.1 M DTT (dithiothreitol) used to reduce disulfide bonds
  • RNase inhibitor (e.g., RNasin Ribonuclease Inhibitor; often 20 to 40 U/μl)
  • M‐MLV Reverse Transcriptase (RNase H)
  • RNase H
  • 0.5‐ml microcentrifuge tubes, RNase‐ and DNase‐free
  • 37° and 65°C incubators
  • 85°C heating block
NOTE: To avoid contamination of samples with RNase, gloves should be worn at all times. Washing of gloved hands with a mild SDS solution (0.01%) can help remove contaminating RNases. Also, micropipettors equipped with barrier‐filter tips should be used when pipetting all solutions. This will prevent the introduction of contaminants located in the chamber of the micropipettors. (Refer to unit 8.2 for a discussion of RNases.)NOTE: Quickly mix the sample by vortex and briefly centrifuge each component for 15 sec at full speed in a benchtop microcentrifuge to collect the samples at the bottom of the reaction tube. This can be done at room temperature.

Basic Protocol 2: Real‐Time PCR Amplification and Analysis

  Materials
  • PCR master mix:
    • 2× reaction mix
    • 50 μM forward primer
    • 50 μM reverse primer
    • RNase‐free water
  • cDNA ( protocol 1)
  • 1.5‐ml microcentrifuge tubes for preparing master mix
  • Thin‐walled PCR tubes
  • Real‐time thermal cycler
NOTE: This protocol describes how to perform a real‐time PCR. When conducting an actual experiment comparing samples, it is important that the amplification efficiencies be known before analyzing the samples. Both housekeeping and target sequences can be prepared using separate master mixes.

Support Protocol 1: Determination of Amplification Efficiency

  Materials
  • cDNA preparation ( protocol 1) or other DNA preparation for amplification efficiency determination
  • PCR master mix:
    • 2× reaction mix
    • 50 μM forward primer
    • 50 μM reverse primer
    • RNase‐free water
  • 1.5‐ml microcentrifuge tubes (for preparing master mix)
  • Thin‐walled PCR tubes
  • Real‐time thermal cycler

Support Protocol 2: Analyzing Results Using the Pfaffl Method to Calculate Fold Induction

  Materials
  • DNA or RNA to be used for standard curve
  • In vitro transcribed RNA (if appropriate)
  • Real‐time thermal cycler
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Figures

Videos

Literature Cited

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  Biéche, I., Olivi, M., Champéme, M.H., Vidaud, D., Lidereau, R., and Vidaud, M. 1998. Novel approach to quantitative polymerase chain reaction using real‐time detection: Application to the detection of gene amplification in breast cancer. Int. J. Cancer 78:661‐666.
  Biéche, I., Laurendeau, I., Tozlu, S., Olivi, M., Vidaud, D., Lidereau, R., and Vidaud, M. 1999. Quantitation of MYC gene expression in sporadic breast tumors with a real‐time reverse transcription‐PCR assay. Cancer Res. 59:2759‐2765.
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  Bustin, S.A. and Nolan, T. 2004. Analysis of mRNA expression by real‐time PCR. In Real‐Time PCR: An Essential Guide (K. Edwards, J. Logan, and N. Saunders, eds.) pp. 125‐184. Horizon Bioscience, Norfolk, U.K.
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  Cheng, J., Zhang, Y., and Li, Q. 2004. Real‐time PCR genotyping using displacing probes. Nucleic Acids Res. 32:e61.
  Chomczynski, P. and Sacchi, N. 1987. Single‐step method of RNA isolation by acid guanidinium thiocyanate‐phenol‐chloroform extraction. Anal. Biochem. 162:156‐159.
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  Eshel, R., Vainas, O., Shpringer, M., and Naparstek, E. 2006. Highly sensitive patient specific real‐time PCR SNP assay for chimerism monitoring after allogenic stem cell transplantation. Lab. Hematol. 12:39‐46.
  Farrell, R.E. 1998. RNA Methodologies: A Laboratory Guide for Isolation and Characterization, 2nd ed. Academic Press, San Diego, Calif.
  Foley, K.P., Leonard, M.W., and Engel, J.D. 1993. Quantitation of RNA using the polymerase chain reaction. Trends Genet. 9:380‐385.
  Gibson, N.J. 2006. The use of real‐time PCR methods in DNA sequence variation analysis. Clin. Chim. Acta 363:32‐47.
  Gibson, U.E., Heid, C.A., and Williams, P.M. 1996. A novel method for real time quantitative RT‐PCR. Genome Res. 6:995‐1001.
  Gudnason, H., Dufva, M., Bang, D.D., and Anders, W. 2007. Comparison of multiple dyes for real‐time PCR: Effects of dye concentration and sequence composition on DNA amplification and melting temperature. Nucleic Acids Res. 35:e127.
  Higuchi, R., Dollinger, G., Walsh, P.S., and Griffith, R. 1992. Simultaneous amplification and detection of specific DNA sequences. Biotechnology 10:413‐417.
  Higuchi, R., Fockler, C., Dollinger, G., and Watson, R. 1993. Kinetic PCR: Real time monitoring of DNA amplification reactions. Biotechnology 11:1026‐1030.
  Kindich, R., Florl, A.R., Jung, V., Engers, R., Müller, M., Schulz, W.A., and Wullich, B. 2005. Application of a modified real‐time PCR technique for relative gene copy number quantification to the determination of the relationship between NKX3.1 loss and MYC gain in prostate cancer. Clin. Chem. 51:649‐652.
  Kochanowski, B. and Reischl, U. 1999. Methods in Molecular Medicine: Quantitative PCR Protocols, 1st ed. Humana Press, Totowa, N.J.
  Königshoff, M., Wilhelm, J., Bohle, R.M., Pingoud, A., and Hahn, M., 2003. HER‐2/neu gene copy number quantified by real‐time PCR: Comparison of gene amplification heterozygosity, and immunohistochemical status in breast cancer tissue. Clin. Chem. 49:219‐229.
  Lee, M.A., Squirrell, D.J., Leslie, D.L., and Brown, T. 2004. Homogenous fluorescent chemistries for real‐time PCR. In Real‐time PCR: An Essential Guide (K. Edwards, J. Logan, and N. Saunders, eds.) pp. 85‐102. Horizon Bioscience, Norfolk, U.K.
  Leutenegger, C.M., Mislin, C.N., Sigrist, B., Ehrengruber, M.U., Hofmann‐Lehmann, R., and Lutz, H. 1999. Quantitative real‐time PCR for the measurement of feline cytokine mRNA Vet. Immunol. Immunopathol. 71:291‐230.
  Liss, B. 2002. Improved quantitative real‐time RT‐PCR for expression profiling of individual cells. Nucleic Acids. Res. 30:e89.
  Liu, W. and Saint, D.A. 2002. Validation of a quantitative method for real time PCR kinetics. Biochem. Biophys. Res. Commun. 294:347‐353.
  Livak, K.J. 2003. SNP genotyping by the 5′‐nuclease reaction. Methods Mol. Biol. 212:129‐147.
  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.
  Logan, J.M.J. and Edwards, K.J. 2004. An overview of real‐time PCR platforms. In Real‐time PCR: An Essential Guide (K. Edwards, J. Logan, and N. Saunders, eds.) pp. 13‐30. Horizon Bioscience, Norfolk, U.K.
  Marras, S.A., Kramer, F.R., and Tyagi, S. 2003. Genotyping SNPs with molecular beacons. Methods Mol. Biol. 212:111‐128.
  Meijerink, J., Mandigers, C., van de Locht, L., Tonnissen, E., Goodsaid, F., and Raemaekers, J. 2001. A novel method to compensate for differential amplification efficiencies between patient DNA samples in quantitative real‐time PCR. J. Mol. Diagn. 3:55‐61.
  O'Garra, A. and Vieira, P. 1992. Polymerase chain reaction for detection of cytokine gene expression. Curr. Opin. Immunol. 4:211‐215.
  Peccoud, J. and Jacob, C. 1998. Statistical estimations of PCR amplification rates. In Gene Quantification (F. Ferre, ed.) pp. 111‐128. Birkhuser, New York.
  Pfaffl, M.W. 2001. A new mathematical model for relative quantification in real‐time RT‐PCR. Nucleic Acids Res. 29:2002‐2007.
  Rasmussen, R. 2001. Quantification on the LightCycler instrument. In Rapid Cycle Real‐time PCR: Methods and Applications (S. Meuer, C. Wittwer, and K. Nakagawara, eds.) pp. 21‐34. Springer, Heidelberg, Germany.
  Temin, H.M. 1974. On the origin of RNA tumor viruses. Ann. Rev. Genet. 8:155‐177.
  Temin, H.M. 1995. Genetics of retroviruses. Ann. N.Y. Acad. Sci. 758:161‐165.
  Tichopad, A., Dilger, M., Schwarz, G., and Pfaffl, M.W. 2003. Standardized determination of real‐time PCR efficiency from a single reaction set‐up. Nucleic Acids Res. 31:e122.
  Valasek, M.A. and Repa, J.J. 2005. The power of real‐time PCR. Adv. Physiol. Educ. 29:151‐159.
  Ward, C.L., Dempsey, M.H., Ring, C.J., Kempson, R.E., Zhang, L., Gor, D., Snowden, B.W., and Tisdale, M. 2004. Design and performance testing of quantitative real time PCR assays for influenza A and B viral load measurement. J. Clin. Virol. 29:179‐188.
Internet Resources
  http://pathmicro.med.sc.edu/pcr/realtime‐home.htm
  University of South Carolina School of Medicine's tutorial on real‐time PCR. At the homepage, click on the “Real‐time PCR tutorial” link under the “Textbook” button.
  http://dna‐9.int‐med.uiowa.edu/
  University of Iowa DNA facility's on‐line tutorial on real‐time PCR. At the homepage, click on the “real‐time PCR” button on the left.
  http://www.lifetechnologies.com/us/en/home/references/ambion‐tech‐support.html
  Ambion's Web site has many valuable technical reports. These can be searched for on the Technical Resources page.
  http://www.gene‐quantification.de/
  A short primer on absolute quantification methods in real‐time RT‐PCR. From the homepage, click on the “RT.gene‐quantification.info.” From the subsequent page, click on the “REVIEW: Absolute quantification of mRNA using real‐time reverse transcription PCR assays.”
  http://www.youtube.com/watch?v=GQOnX1‐SUrI
  This video was created by BioRad and provides a nice overview of data analysis of real‐time PCR results. You can also visit the BioRad Web site for additional real‐time PCR advice at http://www.bio‐rad.com/en‐us/applications‐technologies/qpcr‐real‐time‐pcr.
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