How does RT-PCR work? - (Sep/27/2014 )

This will be a very newb-ish question, so I hope it's okay.  We're doing rt-PCR (reverse transcription PCR, not real time PCR) in my lab, and I'd just like to have a better understanding of how it works.  I've come up with nothing that actually explains the whole process. 

What I'm having the most difficult time with is that (apparently) only two primers are involved: a forward, and reverse. 

I can imagine a scenario in which, say, the reverse primer is responsible for synthesis of mRNA to cDNA. 

But how, then, do we amplify the cDNA in the PCR phase, using only one primer?  It doesn't seem to me that the reverse primer used in the previous step would be able to serve as a primer in cDNA amplification.

To summarize, I can imagine a way of doing this with three unique primers.  One for mRNA -> cDNA, and then two for amplification of cDNA.  If someone could explain this one part, it would help.  Even better would be a link to some resource that explains how the whole thing works.

Thanks!

Actually, after working it out on paper, I do see how the reverse primer from the first step could work as the second primer during cDNA amplification.

So this is the possibility that I've worked out:

In the reverse transcription phase, the reverse primer is used to synthesize the first cDNA strand.

In the PCR phase, the same reverse primer and a forward primer then function in amplification of cDNA. 

Is this correct?  (I'd still love a detailed breakdown of the process if I am.)

We're working with BamHI and EcoRI to insert this cDNA into a plasmid.  Attached is the diagram I worked out, proposing one way the whole thing might work.

Could someone take a look and let me know if this might be pretty close to the truth?

Thanks.

Looks right to me.

Anything I could read that explains RT-PCR?  I really have scoured, but I'm not coming up with much.

Thanks!

Well, there are really two different reactions of importance. The first is a reverse transcription reaction, which uses a single primer to bind to the 3' end of an RNA strand (or in the middle of a longer strand). These reactions operate without cycling, typically at a relatively low temperature, such as 50. This produces a single stranded DNA copy of the RNA strand.

In a second phase, a normal DNA PCR reaction uses the ssDNA strand as a  template, and ampllifies the region between two primers. One of these could be the reverse transcriptase primer, but it need not be.

There are many variations on this. Sometimes the reverse transcriptase and PCR polymerase are both present in a single tube, and both reactions take place without interruption. But it is easier to think about and investigate these two reactions as separate.

Many variations on the reverse transcriptase reaction exist, often involving using modified RNAs as substrates. So, ligating adapters to either or both ends of the RNA strand is common. Also common is the use of a poly-T primer, which binds to the poly-A tail of eukaryotic mRNA transcripts, and allows selective amplification of those, when combined with a gene-speciic primer in the second stage PCR.

You should find a commercial vendor and read their protocols. Qiagen, NEB, Invitrogen all have good kits. The Invitrogen enzymes are likely the gold standard for the RT reaction.