Quantitative RT-PCR : Pitfalls and Potential

Reverse transcription PCR (RT-PCR) represents a sensitive and powerful tool for analyzing RNA. While it has tremendous potential for quantitative applications, a comprehensive knowledge of its technical aspects is required. Successful quantitative RT-PCR involves correction for experimental variations in individual RT and PCR efficiencies. This review addresses the mathematics of RT-PCR, choice of RNA standards (internal vs. external) and quantification strategies (competitive, noncompetitive and kinetic [real-time] amplification). Finally, the discussion turns to practical considerations in experimental design. It is hoped that this review will be appropriate for those undertaking these experiments for the first time or wishing to improve (or validate) a technique in what is frequently a confusing and contradictory field. INTRODUCTION Quantification of steady-state mRNA levels by reverse transcriptionpolymerase chain reaction (RT-PCR) has tremendous potential; however, it is also fraught with significant pitfalls. On the positive side, the technique is exquisitely sensitive, permitting analysis of gene expression from very small amounts of RNA (even at the level of the content of a single cell). Moreover, this approach can be conducted on a large number of samples and/or many different genes in the same experiment. This enables the investigator a measure of flexibility unavailable in more traditional approaches (i.e., Northern blot, dot blot and hybridization protection assays and in situ hybridization). Along with the power and relative ease of quantitative RT-PCR (QRTPCR) come a number of technical hurdles to utilizing the approach in a reliable manner. 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, QRT-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. Since the first reports of QRT-PCR (2,5,18,54), the use of this technique has grown at an exponential rate. Despite this growth, the ability of RT-PCR to accurately quantify mRNA levels is not universally accepted. Additionally, as is typical of most emerging research areas, the terminology and theoretical basis for this technique have become muddled. This review will attempt to clarify the terminology and to describe those elements needed for accurate, precise and reproducible quantification. The basics of RT-PCR will be described first, followed by the mathematical underpinnings of RT-PCR. A subsequent discussion of RNA standard construction and methods of use will lead to considerations of the relative merits of competitive vs. noncompetitive QRTPCR and quantification by real-time monitoring of reaction kinetics. Finally, concerns of RNA isolation, reaction phase, heteroduplex formation and absolute quantification will be reviewed. This presentation is intended to provide the background and theoretical basis for the disparate approaches to QRT-PCR. The overview will assist an investigator in the choice of the best research tools and methods for validating the technique. A number of good reviews and books exist (8,13–15,64,66) that address many of the concerns in quantitative PCR. However, recent developments in QRT-PCR and the special issues of quantifying mRNA (as opposed to DNA) warrant a fresh look at the technique.