RT-PCR

Abstract

The ability to measure RNA levels quantitatively is crucial to the study of gene expression. Northern blots, dot/slot blots, and nuclease protection assays are methods traditionally used for analysis of mRNA expression. These methods, however, require large quantities of RNA and often lack the needed sensitivity. Northern blotting, probably the most widely used method for characterizing RNA, requires 5–10 µg of purified poly(A)-mRNA and has a detection limit of approx 10⁶–10⁷ mRNA copies. Similarly, solution assays, such as RNase protection or S 1 nuclease, require 0.1–1 µg of purified poly(A)-mRNA and have detection limits of approx 10⁵–10⁶ mRNA copies (1). Thus, the required quantities of purified RNA make these methods impractical for many investigators because of the nature of the systems under investigation. For measuring low-abundance transcripts or working with limited amounts of material, the RT-PCR* technique is an excellent alternative to classical blotting and solution hybridization assays (2). RT-PCR couples the tremendous DNA amplification powers of the PCR technique with the ability of RT to reverse transcribe small quantities of total RNA (1 ng or less) into cDNA. Using total cellular RNA rather that purified poly(A)mRNA reduces the possibility of losing messages during the purification process and allows the use of very small quantities of starting material. For example, methods already exist for performing PCR on a single cell (3,4). Other advantages of this technique are its versatility, sensitivity, rapid turnaround time, and the ability to compare multiple samples simultaneously. Although RT-PCR techniques are mostly semiquantitative, progress is being made in the development of several quantitative methods (5).