Total cDNA PCR - (Oct/19/2005 )
Currently working on a project with limited amount of tumor tissue. Extracted RNA and performed RT-PCR to generate cDNAs. Have limited amount, 20 microliters, to use in the charecterization of thousands of candidate genes. Wondering if there is a primer like oligo dt for cDNA that would allow unbiased amplification of total cDNA.
The problem is that there is no 5' primer to use for "amplification" that would bind to all cDNAs I would suggest diluting your cDNA to look at the different candidate genes, theoretically you only need one copy, and you could do nested PCRs or two rounds of ampification...
To determine how much it is feasible to dilute the sample you have to know how much RNA did you use for the first RT, how many cells did the RNA come from... You may not be able to analyze "thousands" of different genes depending on how much sample you have
If you can start over at the RT step I think there are protocols where you could link an adapter primer to the 5' end of all the cDNAs during synthesis and then you could potentially amplify them; although some mRNAs are very long which may still result in biased amplification (of small mRNAs)... Maybe someone knows a way this can be done with cDNAs...
also look at protocols for amplifying samples for microarrays for suggestions
I hope these suggestions help, good luck!
make a cDNA library, if the 20ul is all you got to work with
I can't think of anything else that will allow you to characterize a whole bunch of genes
this is method of amplification, not using PCR
amikins is right cDNA library is the way to go...