Reverse Transcription Polymerase Chain Reaction - History

History

Since its introduction in 1977, Northern blot had been used extensively for RNA quantification despite its shortcomings of: (a) being time-consuming, (b) requiring a large quantity of RNA for detection, and (c) being quantitatively inaccurate in the low abundance of RNA content. However, the discovery of reverse transcriptase during the study of viral replication of genetic material led to the development of RT-PCR which has since displaced Northern blot as the method of choice for RNA detection and quantification.

RT-PCR has risen to become the benchmark technology for the detection and/or comparison of RNA levels for several reasons: (a) it does not require post PCR processing, (b) a wide range (>10^7 fold) of RNA abundance can be measured, and (c) it provides insight into both qualitative and quantitative data. Due to its simplicity, specificity and sensitivity, RT-PCR is used in a wide range of applications from experiments as simple as quantification of yeast cells in wine to more complex uses as diagnostic tools for detecting infectious agents such as the avian flu virus.

However, RT-PCR is not without a major flaw of its own. The exponential growth of the reverse transcribed complementary DNA (cDNA) during the multiple cycles of PCR produces inaccurate end point quantification due to the difficulty in maintaining linearity. In order to provide accurate detection and quantification of RNA content in a sample, qRT-PCR was developed using fluorescence-based modification to monitor the amplification products during each cycle of PCR.