RT-PCR

http://www.bio.davidson.edu/courses/immunology/Flash/RT_PCR.html
1. uses reverse transcriptase to reverse transcribe the mrna exons to cdna
2. use taq polymerase and primers to transcribe

http://pathmicro.med.sc.edu/pcr/realtime-home.htm

1. mRNA is copied to cDNA by reverse transcriptase using an oligo dT primer (random oligomers may also be used). In real-time PCR, we usually use a reverse transcriptase that has an endo H activity. This removes the mRNA allowing the second strand of DNA to be formed. A PCR mix is then set up which includes a heat-stable polymerase (such as Taq polymerase), specific primers for the gene of interest, deoxynucleotides and a suitable buffer.
pp-pcr2.gif (9025 bytes) pp-pcr3.gif (7124 bytes) 2. cDNA is denatured at more than 90 degrees (~94 degrees) so that the two strands separate. The sample is cooled to 50 to 60 degrees and specific primers are annealed that are complementary to a site on each strand. The primers sites may be up to 600 bases apart but are often about 100 bases apart, especially when real-time PCR is used.

3. The temperature is raised to 72 degrees and the heat-stable Taq DNA polymerase extends the DNA from the primers. Now we have four cDNA strands (from the original two). These are denatured again at approximately 94 degrees.