Single Worm RT-PCR woes

[Glenn White]

Howdy:

We have been having a terrible time with our attempts at single worm 
RT-PCR.  We've encountered every possible
problem, and now are dealing with the fact that we cannot seem to 
prevent degradation of the mRNA before we
amplify it (even though we have gotten amplication of a cDNA before, 
but only sporatically).

We're following the digestion with Proteinase K for 60 min @ 65 oC, 
followed by a kill step, then add the rxn
mix and amplify.

We keep getting amplification of the genomic fragment (primers span 
an intron).  We're using actin primers
right now.

If any of you have had some similar problems and solved them, I'd be 
most appreciative of hearing from you.

We have to do single worm RT, a total RNA prep from a large 
population of animals will not suit our needs.

As always, if I get enough responses, I will summarize them to the 
entire group.

If you are willing to send me your protocol, I'd be grateful for that too.

Cheers,

Glenn

Dr. Glenn Edward White
Academic Honors Director
Associate Professor of Biology
Ashland University
Ashland, Ohio 44805
419-289-5259
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