I concur with Joeri's response and add the following. Read them very closely! There is far more in them than it appears. I have been using RT-PCR to look at rare messages in primary peritoneal mesothelial cells. I have found that the commonly-used beta-Actin, Histone and GAPDH primer/probes vary with cell cycle and culture status (ie log versus stationary, passage). Not surprising when you think it through it. Thus I am sceptical about much of the published literature. Ideally, you need an invariant control message of approximately equivalent expression to your target. Of course, I design my primers to span intron-exon boundaries, but sometimes these are not known. In this event I choose the forward primer in a region of the message that is upstream, within in the prepro- or signal-peptide coding region. I also design primers to generate products of similar size for both target and control (eg 400-500 bp) so that post-electrophoresis staining is semi-quantitative. I use GAPDH, but have primer designs for pyruvate dehydrogenase and histone 3A. One potential problem in GAPDH is the presence of pseudogenes although I have not personally experienced this problem. We do not have a 7700 so one work-around we have used with partial success has been the Ambion competimer strategy. About equivalent to our success with competitive PCR (not good enough). However that took considerable fiddling to get it right (which Ambion blamed on the rarity of our target message) Beta 2 microglobulin was recently suggested to me on the basis that ALL cells express it (even my meso/epithelial cells where it too is a rare message). If you'd like my primer designs or more on my success and failures email me. Cheers David L.
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