Quantitative Measurement of mRNA Expression by Competitive RT-PCR

As a method of specific mRNA detection, the single most important advantage of RT-PCR is its sensitivity, because of the remarkable sensitivity of PCR. Isolation of polyA⁺ mRNA is unnecessary, and minute amounts of total RNA suffice. If random primers are used in the reverse transcription reaction, a single cDNA preparation can be used for the detection of numerous different mRNAs in the same RNA sample. This sensitivity means that RT-PCR is a powerful technique, particularly when tissue availability is limiting, or when the mRNA to be detected is present in low abundance. We have routinely detected low abundance mRNAs for various cytokine receptors in RNA samples isolated from small pinch biopsies of human colonic mucosal epithelium. The technique has also been used for the sensitive detection of viral RNA, such as from HIV and HCV, in human serum, where the viral titer is frequently <1000 virus particles per milliliter of serum.