| RT-PCR: The Basics RT-PCR (reverse transcription-polymerase chain reaction) is the most sensitive technique for mRNA detection and quantitation currently available. Although this discussion will focus on RT-PCR as a method of RNA quantitation, RT-PCR can also be used for cloning, cDNA library construction, probe synthesis, differential display, and signal amplification in in situ hybridizations. The technique consists of two parts: synthesis of cDNA from RNA by reverse transcription (RT) and amplification of a specific cDNA by polymerase chain reaction (PCR). For a comparison of mRNA quantitation methods see "Strategies for Detecting mRNA". To view an animated tutorial on RT-PCR, including a discussion of quantitation strategies, see "RT-PCR Problem Solver" (requires Flash plug-in). The first consideration when using RT-PCR for mRNA analysis is RNA isolation. The RNA should be high quality and free from genomic DNA contamination. However, since most RT-PCR methods amplify only a few hundred bases rather than the complete mRNA sequence, the sample RNA can be slightly degraded. For more information, see the articles, "Avoiding DNA Contamination in RT-PCR", "Methods to Remove DNA Contamination in RT-PCR" and "RT-PCR Without Prior RNA Isolation". Every RT-PCR begins with a reverse transcriptase reaction. The RT reaction uses an RNA template (typically either a total or poly(A) RNA sample), a primer (random or oligo dT primers), rNTPs, buffer and a reverse transcriptase (M-MLV or AMV RT). For additional information, see "How to Choose a Kit for Reverse Transcription". With use of Ambion's RETROscript™ Kit, the RT reaction requires little optimization, yet provides flexibility. RETROscript contains both oligo(dT) and random primers, and can be used in a one- or two-step reaction format. The RT reaction is incubated for 1 hour at 42°C and generates a single-stranded DNA molecule complementary to the RNA (cDNA). The cDNA serves as a template in the PCR reaction. Components of the PCR, in addition to the cDNA, include dNTPs, buffer, thermostable DNA polymerase and primers specific for the gene of interest (gene specific primers). The cDNA is amplified exponentially via cycles of denaturation, annealing and extension. Because amplification is exponential, small sample-to-sample concentration and loading differences are amplified as well; therefore, PCR requires careful optimization when used for quantitative mRNA analysis. Relative vs. Competitive vs. Comparative RT-PCR Like other methods of mRNA analysis, such as Northern blots and nuclease protection assays, RT-PCR can be used for relative or absolute quantitation. Relative quantitation compares transcript abundance across multiple samples, using a co-amplified internal control for sample normalization. Results are expressed as ratios of the gene specific signal to the internal control signal. This yields a corrected relative value for the gene specific product in each sample. These values may be compared between samples for an estimate of the relative expression of target RNA in the samples; for example, 2.5-fold more IL-12 in sample 2 than in sample 1. Absolute quantitation, using competitive RT-PCR, measures the absolute amount (e.g., 5.3 x 105 copies) of a specific mRNA sequence in a sample. Dilutions of a synthetic RNA (identical in sequence, but slightly shorter than the endogenous target) are added to sample RNA replicates and are co-amplified with the endogenous target. The PCR product from the endogenous transcript is then compared to the concentration curve created by the synthetic "competitor RNA." Comparative RT-PCR mimics competitive RT-PCR in that target message from each RNA sample competes for amplification reagents within a single reaction, making the technique reliably quantitative. Because the cDNA from both samples have the same PCR primer binding site, one sample acts as a competitor for the other, making it unnecessary to synthesize a competitor RNA sequence. Both relative and competitive RT-PCR quantitation techniques require pilot experiments. In the case of relative RT-PCR, pilot experiments include selection of a quantitation method and determination of the exponential range of amplification for each mRNA under study. For competitive RT-PCR, a synthetic RNA competitor transcript must be synthesized and used in pilot experiments to determine the appropriate range for the standard curve. Comparative RT-PCR yields similar sensitivity as relative and competitive RT-PCR, but requires significantly less optimization and does not require synthesis of a competitor. |