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

  •   FigureFigure 10.3.1 Comparison of endpoint RT‐PCR and real‐time RT‐PCR. Both procedures begin with isolation of RNA followed by characterization for purity and integrity. Purified RNA is then used as template to generate first strand cDNA. During endpoint PCR, DNA is measured at the completion of PCR amplification. Quantification of DNA product is determined by gel electrophoresis, staining of separated DNA fragments with a fluorescent dye, and digital imaging densitometry to measure DNA band intensity. In real‐time PCR, DNA is measured during the exponential phase of PCR amplification. Accumulating product is detected as it is being amplified using fluorescent DNA probes. Both endpoint PCR and real‐time PCR data analysis require normalization of data to known standards to determine relative or absolute quantity of starting target gene expression.
  •   FigureFigure 10.3.2 Theoretical doubling and experimental accumulation of target DNA during PCR. Starting with one molecule, the table in (A) shows DNA accumulation during each cycle under theoretical doubling conditions (diamonds), or as might be seen in an experimental assay (squares). The data in table (A) are plotted as a linear graph (B) and as a logarithmic graph (C). x axis: cycle number; y axis in (B): molecules of DNA; y axis in (C): log10 [molecules of DNA].
  •   FigureFigure 10.3.3 Plot of an experimental PCR reaction performed on a BioRad iQ5 real‐time PCR instrument with duplicates of each sample. x axis: cycle number; y axis: amount of DNA. Notice that the three PCR phases—exponential, linear, and plateau—vary for the four samples.
  •   FigureFigure 10.3.4 Schematic drawing showing the steps of reverse transcription PCR reaction. mRNA is depicted as a gray line, while DNA is black. RT: reverse transcriptase; Taq: thermostable DNA polymerase. In the first step, RT transcribes a DNA copy of the message RNA using a poly‐dT primer. Gene‐specific primers are used to amplify specific targets using Taq polymerase, as shown in the figure.
  •   FigureFigure 10.3.5 Flow chart of a real‐time PCR experiment. The first step is to determine how to optimally isolate RNA and create the cDNA. Once a suitable cDNA template is created, it will be necessary to optimize the PCR reaction. The most critical aspects to consider when optimizing a PCR reaction are the annealing temperature and the MgCl2 concentration. Other factors may be important if experiencing problems in generating a specific PCR product. After appropriate PCR conditions are determined, the amplification efficiency should be determined so that accurate comparisons can be made.
  •   FigureFigure 10.3.6 Designing primers for real‐time RT‐PCR. Ideally, primers will be designed to straddle an intron such that it can anneal only to the cDNA synthesized from the spliced message RNA, as shown on the left. Any contaminating genomic DNA would not be suitable for primer annealing. Alternatively, primers can be designed to flank a large intron, and only cDNA synthesized from a spliced message RNA would efficiently produce a PCR product, as shown on the right.
  •   FigureFigure 10.3.7 Real‐time cyclers. Examples of two types of real‐time thermal cyclers. (A) and (B), Roche LightCycler. In this system the reactions are set up in borosilicate 20‐μl capillaries, and the circular “rotor” accommodates up to 32 reactions at one time. (C) and (D), BioRad iQ5, which uses 96‐well reaction format; up to five targets can be multiplexed in one well.
  •   FigureFigure 10.3.8 SYBR Green during PCR amplification: the fluorescence of this dye increases 100‐ to 200‐fold when bound to the minor groove of double‐stranded DNA. This is used to measure the amount of DNA at the end of the elongation step of the PCR reaction.
  •   FigureFigure 10.3.9 Optical design for detection of fluorescence in a real‐time PCR thermal cycler. The detection system of the real‐time thermal cycler is a excitation and emission filter‐based system. Depending on the instrument manufacturer, the light source consists of a xenon, tungsten‐halogen, or LED (light emitting diode) light, and the detector is a CCD (charge‐coupled device) camera or PMT (photomultiplier tube). See UNIT for further discussion.
  •   FigureFigure 10.3.10 TaqMan probe used for sequence‐specific amplification of DNA fragments. A laser excites the reporter fluorochrome and it then emits a light that is absorbed by the nearby quencher. After the 5′ nuclease activity of Taq polymerase, the quencher is no longer in close proximity to the reporter, and thus the light emitted by the reporter can be detected. R: reporter; Q: quencher (adapted from Valasek and Repa, ).
  •   FigureFigure 10.3.11 Melting curve analysis performed on the BioRad iQ5 real‐time PCXR instrument. Panel (A) shows double peaks. The lower peaks represent DNA fragment denaturing at lower temperature, most likely due to primer‐dimers. Panel (B) shows a single peak melting curve, representative of a single species of DNA molecule in the reaction.
  •   FigureFigure 10.3.12 Setting the threshold. Example of an experimental amplification performed on the BioRad iQ5 real‐time PCR instrument: (A) linear plot of the amount of DNA ( y axis) and the cycle number ( x axis); (B) logarithmic plot. The threshold line is used to quantify the amount of DNA and is set at the exponential phase of the amplification. CT: cycle threshold.
  •   FigureFigure 10.3.13 Effect of the PCR amplification efficiency on the accumulation of the DNA product. (A) The table shows values for 100%, 90%, 80%, and 60% efficient accumulation of product per cycle starting with 1 molecule. (B) and (C) are the linear and logarithmic plots respectively of values in the table.
  •   FigureFigure 10.3.14 Standard curve analysis performed on the BioRad iQ5 real‐time PCR instrument. Panel A: linear plot; Panel B: logarithmic plot; Panel C: standard curve. Five dilutions of a sample were run in triplicate to determine the melting curve (circles). The concentration of DNA in the unknown samples, also run in triplicate, was determined by plotting the CT values on the curve (crosses).

Videos

Literature Cited

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  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.
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  Liss, B. 2002. Improved quantitative real‐time RT‐PCR for expression profiling of individual cells. Nucleic Acids. Res. 30:e89.
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  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.
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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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