rna isolation manually - (Jun/22/2010 )
I am doing rna isolation manually by the phenol/sds method from plant source . After running the rna on agarose denaturing gel i got 2 intact bands of the rRNA , then prepared c-DNA and tried to amplify with housekeeping gene primer but i didnt get any amplification. I had stored the rna in -70C for 1 day before cDNA preparation. Is this why i didnt get any amplification. Does this m-rna in the whole rna isolation process degrade so fastly?
Please help me with some suggestions
this storage step is no problem. I guess something went wrong during the cDNA synthesis or the PCR.
What species of plant did you use? Does your lysis buffer have a strong reducing agent (such as DTT or beta-mercaptoethanol)? Some plants are high in compounds that can modify RNA or otherwise inhibit the RT reaction. If phenol isn't completely removed in the chloroform extraction step, this can also inhibit the RT reaction.