Real-time PCR Overview (Word).doc


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Real-time PCR Overview (Word).doc

  1. 1. Real-Time PCR Overview Topics covered in this document: 1. What is real-time PCR 2. Chemistries used in real-time PCR 3. General considerations (application note from Bio-Rad) 4. Getting started 5. Probe design 6. Suggested papers to read Section 1. What is real-time PCR and what is it used for? Real-time PCR has the ability to directly measure the PCR reaction as amplification is taking place with the use of fluorescent molecules. Basically, you add something to your PCR reaction that will keep fluorescing brighter and brighter with the more PCR product that is produced in the sample well. Real-time PCR is used to quantitate DNA or RNA samples. The following is a short list of experiments that can use real-time PCR for quantitation: 1. Gene expression-replacing northern blots, competitive PCR, differential display, RNase protection assays 2. Viral load 3. GMO testing (genetic modified organism) 4. Validating micro arrays 5. ChIp-Chromatin Immunoprecipitation 6. Transgenic screening Why do real-time PCR if there are other tools and protocols already established? 1. Real-time PCR is fast. A typical real-time PCR run takes 90 minutes to complete. How many days does it take to do a northern blot? 2. Real-time PCR is sensitive. Your iCycler iQ can detect a 2-fold difference between samples. 3. Large dynamic range. The iQ can detect quantities from 109 down to a single copy. 4. Multiplexing capability. The iQ can amplify and detect up to 4 independent targets in a single well. 5. No radioactivity 6. No post run manipulations of samples-no gels, blotting or imaging 7. Typically use less starting material than other tools. Why would we want to use real-time PCR to quantitate our samples? PCR amplification is exponential. Exponential amplification means that there is a doubling of the amount of PCR product that is produced at the completion of each cycle. All samples should be amplifying with the same efficiency during this part of amplification. This exponential amplification is the most efficient part of amplification and does not occur indefinitely due to the competition between single stranded PCR product and primers to bind complementary sequence and also the availability of dNTPs, primers, and enzyme. Following the exponential increase of PCR product is a linear increase and finally a plateau. The efficiencies of amplification in the linear and plateau phase will vary, even among identical samples. This means that end-point measurements are not very accurate and to generate a standard curve using final products produced would probably not produce valid data. Real-time PCR actually allows us to use the data that is collected during exponential amplification (when all samples are amplifying at the same efficiency) to set up a standard curve or normalize values against a reference sample. What values are used in real-time PCR? Ct or threshold crossing values are assigned to each sample after a real-time PCR run is completed. The Ct is the exact cycle that crosses the threshold. The threshold is a horizontal line drawn through the amplification plot. This is drawn at the RFU (relative fluorescent unit) where there is a significant increase of fluorescence above background fluorescence. Basically this threshold line is drawn somewhere in the exponential amplification of your samples. The iCycler iQ will make an automatic threshold call for you (if desired, you can adjust this manually as well).
  2. 2. Reality vs. Theory of PCR amplification: Amplification is exponential, but the exponential increase is limited: -Linear increase follows exponential amplification -Eventually plateaus Theory Reality Log target DNA Plateau Linear Exponential Cycle # 96 replicates with different end point values Threshold line Ct
  3. 3. Section 2. Chemistries used in real-time PCR 1. non-specific DNA binding dyes: SYBR green/ethidium bromide These dyes will fluoresce much more brightly when bound to double-stranded DNA than when they are in solution. The more PCR product that is produced, the more DNA binding dye will bind and then the higher the fluorescene. Two drawbacks to using the DNA binding dyes are that they will fluoresce when bound to any dsDNA (primer dimers and non-specific products) and also you can not multiplex with them. SYBR green step process: 1. At the denaturation step, all SYBR green is free in solution fluoresceing with a low level of back ground fluoresence. 2. During the annealing step the primers anneal to their complementary sequence. As soon as this occurs, Taq will begin the extension step generating double-stranded DNA. 3. SYBR green will not bind to this double-stranded DNA. It is at this step that we excite our samples and read the fluorescence that is emitted. So the more PCR product that is present in the well, the higher the fluoresce signal is generated in that well. DNA binding dyes BD 3’ 5’ BD 5’ 3’ 3’ 5’ Annealing 5’ 3’ BD BD Extension 3’ BD Taq 5’ 5’ BD 5’ 5’ BD BD BD 3’ Taq Extension Continued Apply Excitation λ λ λ Taq 5’ Wavelength 3’ 5’ BD BD BD 5’ BD BD Taq 3’ λ λ Repeat
  4. 4. 2. Hybridization probes (Taqman/hydrolysis probes, Molecular Beacons, FRET, Scorpions/amplifluors) These types of probes are specific to your PCR product and the fluorescence will increase as the amount of PCR product in the well increase. You can multiplex with these type of probes. Taqman/hydrolysis probes step process: 1. A linear oligonucleotide probe is constructed that has a 5’ fluorescent dye reporter and a 3’ quencher. While the probe is intact if the 5’ reporter is excited, its energy is transfred to the 3’ quencher. This will deminish any fluorescence from the reporter dye. 2. If a complemtary sequence is present in the well (a target sequence), the probe will anneal to that sequence. The probe is designed to anneal about 7-10 C above the annealing temperature of the primers. 3. Once the primers have annealed, Taq will begin its extension step from the 3’ end of the primers. Taq will cleave the Taqman probe when it incounters that probe. 4. The reporter dye is now free to fluoresce because the reporter dye and quencher are not longer in close proximity to each other. The more target sequences present, the more probes are cleaved and the higher the fluorescence signal generated. Cleavage-based assay: TaqManTM Q R 5’ 3’ 5’ 3’ 5’ 3’ Annealing R Extension Step Q R Taq 3’ 5’ 1. Strand Displacement 5’ 3’ R Q Taq 5’ 3’ 5’ 3’ 2. Cleavage R Taq Q 3’ 5’ 3. Polymerization Complete 5’ 3’ λ R Taq Q 5’ 3’ 5’ 3’ 4. Detection Section 3. Select Application Notes from Bio-Rad: Tech Note 2567: “Real-Time PCR Using the iCycler iQ Detection System and Intercalation Dyes” Tech Note 2568: “iCycler iQ Detection System for TaqMan Assays” Tech Note 2593: “Real-Time PCR General Considerations” Tech Note 2679: “Real-Time Multiplex PCR from Genomic DNA Using the iCycler iQ Detection System” Tech Note 2696: “Multiplex Relative Gene Expression Analysis by Real-Time RT-PCR Using the iCycler iQ Detection System” These application notes and several others can be viewed and/or downloaded from the Bio-Rad website: • From the Bio-Rad home page, select “Life Science Research” from the Online Catalog section on the left side of the page. Then select the “Amplification Products” link on the right hand side of the page. • Select the link for the iCycler iQ system, and then click on the “Bibliography” link. • The Bibliography lists publications from other scientists using the iQ system as well as the application notes from Bio-Rad’s Research and Development team.
  5. 5. Section 4. Getting started with real-time PCR 1. Amplicon Design: Amplicon (PCR product) 75-150 bp in length. The smaller the amplicon usually the more efficient the PCR reaction. Real-time PCR reactions need to be as efficient as possible. Reactions with poor efficiency will produce inconsistent and varied results. Design and order primers. Primers need to be desalted and do not need any extra purification. 2. PCR dilution series to evaluate primer set efficiency with SYBR Green I By performing a serial dilution of a template (gene cloned into a plasmid, cDNA, or genomic DNA), the efficiency of the amplification reaction is determined. During exponential amplification, 2n=dilution factor, where n is equal to the difference in Ct values between sequential dilutions. The Ct value differences in a 2-fold serial dilution will be 1 cycle for a 100% efficient reaction. (10-fold will be 3.3 cycle differences) The efficiency can give you valuable information about the chemistry of your reaction. The efficiency (overall amplification rate) of a primer pair is determined from the slope of a standard curve the primers generate with a given target sequence. The current version of the iQ software automatically converts the slope of the standard curve to a percentage rating of reaction efficiency, which is displayed as part of the dataset: Efficiency = [10(-1/slope)] - 1 For example: a slope of 3.322 ~ 100%, a slope of 3.5 ~ 95%. At 100% efficiency the template doubles after each cycle during exponential amplification. Several design factors influence efficiency such as the length of the amplicon, the G/C content of the amplicon and secondary structure. The dynamics of the reaction itself can also influence efficiency. Variations in the dynamics can result from such sources as the enzymes used in the reaction and non-optimal reagent concentrations. 3. Check for primer set specificity Use melt curve and then confirm with gel. Run at least one reaction on a gel when you are working with a new set of primers to confirm that the correct size PCR product was made. 4. Design and order specific probe, if desired 5. PCR dilution series to evaluate hybridization probe reaction efficiency Confirm probe behavior over wide dynamic range (5 to 6 orders of magnitude) by running a dilution series of the template, and examining the resulting PCR efficiency and Correlation Coefficient. Section 5. Probe and primer design Primer design: • Targets an amplicon length of 75 to 150 bp • Melting Temperature (Tm) above 50 oC • 50 to 60% GC content (this is driven mainly by the Tm • Limit secondary structure • Limit stretch of G or C’s longer than 3 bases • No stable interaction between forward and reverse primers (primer/dimer pairs)
  6. 6. • Place C’s and G’s on ends of primers, but no more than 2 in the last 5 bases on 3’ end Verify specificity, use the BLAST website for this: Poor amplification due to primer dimers or non-specific amplification can occur. Eliminate primer dimers by trying one or more of the following: Titrate down concentration of primers (optimal primer concentrations are typically between 100 – 500 nM each primer) Adjusting annealing temperature (use gradient real-time PCR and look for the temperature that yields the earliest Ct) Redesigning one or both primers Taqman probes should have a minimum of secondary structure to work well. No G at 5’ and length of the probe. These types of probes are not as well quenched when the probe itself gets long (over 24 bp). Section 6. Suggested review papers to read These are a few papers that are good reviews of what real-time PCR is and also general considerations. Absolute quantification of mRNA using real-time reverse transcription polymerize chain reaction assays. Bustin, SA. Journal of Molecular Endocrinology (2000) 25, 169-193. Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): trends and problems. Bustin, SA. Journal of Molecular Endocrinology (2002) 29, 23-29. Real-time PCR in virology. Mackay, Ian M. Nucleic Acids Research (2002) Vol. 30, No.6 pp 1292-1305. An Overview of Real-time quantitative PCR: Applications to Quantify Cytokine Gene Expression. Giulietti, Annapaula. Methods. (2001) 25, 386-401. Housekeeping Genes Schmittgen TD, Zakrajsek BA. Effect of experimental treatment on housekeeping gene expression: validation by real-time, quantitative RT-PCR. J Biochem Biophys Methods. 2000 Nov 20; 46(1-2): p69-81. Thellin O, Zorzi W, Lakaye B, De Borman B, Coumans B, Hennen G, Grisar T, Igout A, Heinen E. Housekeeping genes as internal standards: use and limits. J Biotechnol. 1999 Oct 8; 75(2-3): 291-5. Suzuki T, Higgins PJ, Crawford DR. Control selection for RNA quantitation. Biotechniques. 2000 Aug; 29(2): 332-7. Review. Bustin SA. Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. J Mol Endocrinol. 2000 Oct;25(2):169-93. Review. Hamalainen HK, Tubman JC, Vikman S, Kyrola T, Ylikoski E, Warrington JA, Lahesmaa R. Identification and validation of endogenous reference genes for expression profiling of T helper cell differentiation by quantitative real-time RT-PCR. Anal Biochem. 2001 Dec 1;299(1):63-70.
  7. 7. Vandecasteele SJ, Peetermans WE, Merckx R, Van Eldere J. Quantification of expression of Staphylococcus epidermidis housekeeping genes with Taqman quantitative PCR during in vitro growth and under different conditions. J Bacteriol. 2001 Dec;183(24):7094-101. Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, Speleman F. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes Genome Biology 2002 3(7): research 1-11 Selvey S, Thompson EW, Matthaei K, Lea RA, Irving MG, Griffiths LR. Beta-actin--an unsuitable internal control for RT- PCR. Mol Cell Probes 2001 Oct;15(5):307-11 Tricarico C, Pinzani P, Bianchi S, Paglierani M, Distante V, Pazzagli M, Bustin SA, Orlando C. Quantitative real-time reverse transcription polymerase chain reaction: normalization to rRNA or single housekeeping genes is inappropriate for human tissue biopsies. Anal Biochem. 2002 Oct 15;309(2):293-300. Relative Expression Calculations Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 2001 Dec;25(4):402-8. Pfaffl MW. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res. 2001 May 1;29(9):e45. ABSOLUTE STANDARD CURVE VS RELATIVE STANDARD CURVE VS COMPARATIVE CT METHOD Johnson MR, Wang K, Smith JB, Heslin MJ, Diasio RB. Quantitation of dihydropyrimidine dehydrogenase expression by real-time reverse transcription polymerase chain reaction. Anal Biochem. 2000 Feb 15;278(2):175-84. ABSOLUTE QUANTITATION Li X, Wang X. Application of real-time polymerase chain reaction for the quantitation of interleukin-1beta mRNA upregulation in brain ischemic tolerance. Brain Res Brain Res Protoc. 2000 Apr;5(2):211-7. RELATIVE QUANTITATION - STANDARD CURVE VS COMPARATIVE CT METHOD Winer J, Jung CK, Shackel I, Williams PM. Development and validation of real-time quantitative reverse transcriptase-polymerase chain reaction for monitoring gene expression in
  8. 8. cardiac myocytes in vitro. Anal Biochem. 1999 May 15;270(1):41-9.