RNA extraction, RT-PCR problems

Austin P. So (Hae Jin) nobody at nowhere.com
Mon Apr 24 22:41:06 EST 2006


Jen Han wrote:

> For any given batch of samples, we always experience a few samples that give
> no amplification product in the RT-PCR despite equal handling of all samples
> during RNA isolation, reverse transcription, and RT-PCR.  Initially, we
> assumed error during the RT-PCR procedure and ran another plate with all
> those samples, but the same samples that were bad the first time yielded no
> product again.  Then, we wondered if certain of the cDNA samples were bad
> because of error during the reverse transcription procedure, so we re-did
> the RT with all samples and ran another RT-PCR with the newly made cDNA.
> Again, the same samples yielded no product.  Thus, it must mean that for
> these samples, the RNA was bad in the first place...

I don't know if this will help, but it may not necessarily be your RNA:

1. when you say "samples" do you mean that you are interrogating the 
same batch of cDNA with different TaqMan probes? Do some consistently 
work as well as consistently not work? Do you run the PCR reaction out 
on a gel to see if there really isn't any product?

2. how old are your probes? how were they aliquoted? how were they designed?

3. did you optimize your PCR before you performed you taqman assay?

You might also want to run this by the qpcrlistserv in Yahoo groups:
http://groups.yahoo.com/group/qpcrlistserver/?yguid=175253511

Cheers

Austin


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