Amplification of region from sperm RNA - sperm RNA stability (Dec/28/2010 )

I have this weird problem, i take swimup of the human spermatozoa. Then extract RNA using Trizol method, i get a very low yield, somewhere around 22ug/ml. This I use it as an initial template and amplify the product of around 1.9kb. When my RNA is Fresh i get a very faint band in the gel. But after a weeks time if I try to amplify from the same batch of RNA I dont get the band. This hs been consistently happening and very disappointing. Currently I was storing my RNA @ -20deg. What would be the problem? can anyone tell me. I am using a Onestep RT PCR Kit containing AMV RT and Hi-Fidelity Taq Polymerase.

RNA is more stable at -70. Try storing at -70. Avoid multiple freeze-thaws. hope this was helpful

Do two steps. Use any reverse transcriptase and DNA polymerase that works for you.
And cDNA is better for storage than RNA.

Another thing, if you know the sequence of the region you want to amplify, design specific reverse primer, instead of polyT.

kajmak on Fri Dec 31 08:08:37 2010 said: