Fig. 9. Reverse Transcriptase coupled to PCR. One of the most important uses of the PCR is to measure the amounts of a specific mRNA. Changes in the accumulated levels of a specific mRNA can be highly diagnostic of disease processes, for example in the prognosis of breast cancer.20 To accurately and precisely measure the level of a specific mRNA, total RNA from a tissue is copied into cDNA with reverse transcriptase and a specific primer (Primer 1 in the illustration). A second specific primer (Primer 2) is used to initiate elongation of the cDNA with Taq polymerase or a related temperature-stable polymerase. A double-stranded cDNA results from these two steps, and the same primer pair is used to continue the PCR. Provided that the reaction is not cycled so many times that the substrates are depleted, the amount of PCR products is proportional to the starting concentration of the mRNA of interest. By carrying out simultaneous reactions with standard amounts of the specific mRNA, a calibration curve can be generated to calculate the absolute amounts of starting mRNA. This allows precise measurements of the changes in mRNAs in response to drug treatments, experimental manipulations, or disease courses.183