Reverse Transcriptase PCR (Not to be confused with Real Time PCR) in comparison to traditional approaches (i.e., Northern blot, dot blot and hybridization protection assays and in situ hybridization) RT-PCR-based assays are the most common method for characterizing or confirming gene expression patterns and comparing mRNA levels in different sample populations.
Since the first reports of quantitative RT-PCR , the use of this technique has grown at an exponential rate. Despite the power and relative ease growth, the ability of RT-PCR to accurately quantify mRNA levels is hampered by a technical hurdle. Specifically, as the PCR greatly amplifies the target, errors are also amplified.
As a result, variability can be very large and preclude accuracy and reliable quantification. For this reason, quantitative RT-PCR is viewed with skepticism by many investigators and reviewers. Such concern can be obviated by thoughtful experimental design and carefully validating the technique for a given gene or a given laboratory. See PCR product on Offers for Labs