RT-PCR mouse RNA released from complement-lysed cells - Goal: to whether RNA is of nuclear or mitochondrial origin. Planning to PCR ampl (Apr/26/2006 )

Dear all,

I'm trying to determine the origin of RNA molecules released from mouse T-cell lymphoma cells following complement-mediated lysis.

I've killed the cells using complement. Released nucleic acids have been isolated by low speed centrifugation of the killed cells, and purified using a Roche kit.

My original plan was based on DNA. If the non-descript nucleic acid released were DNA, then I would simply PCR amplify using primers specific for nuclear (i.e. GAPDH) vs. mitochondrial genomes (i.e. mitochondrial rRNA).

However, the majority of the nucleic acid is RNA, which is still within my interest. Thus, I will need to reverse transcriptase PCR the non-specific RNA, and then proceed with the PCR experiment on the cDNA.

The non-specific RNA was separated by agarose gel electrophoresis and there was clear apoptotic laddering as well as the two ribosomal RNA subunits.

My question is what are the best primers to use for the RT-PCR.

Thank you very much,
Sincerely,

Tony, undergraduate
Duke University
DNA immunology

My advice would be to purchase a RT kit. They will include Universal primers, enzyme and buffer. This will ensure that the primers and enzyme are completely compatible and you get all components, ready to go for each reaction.

Alternatively, you can find commercial primers seperately and use whatever RT enzyme you prefer from any supplier - NEB, Invitrogen, ABI, Promega etc.