Beta-Actin good RT-PCR Control?
Richard R. Hardy
hardy at mighty.fccc.edu
Fri Dec 30 20:15:25 EST 1994
In article <smh1008-311294010018 at crcmac2.path.cam.ac.uk>,
smh1008 at cus.cam.ac.uk (David Huen) wrote:
> This might be a bit of a dumb question but it has puzzled me for a long
> time. How do you use beta-actin as an RT-PCR control when genomic DNA has
> beta-actin pseudogenes (it does, doesn't it?)? Surely under these
> circumstances, irrespective of the integrity of your mRNA and success of
> your RT reaction, there should be enough genomic DNA carryover to at least
> occasionally generate a false positive in RT-PCR.
> David Huen aka Puzzled, Dept. of Pathology, Univ. of Cambridge.
Dear Puzzled in Cambridge,
Well, with our particular b-actin primers (this is in mouse), we
see a band about 70-100 nt larger than the c-DNA band if there is
contaminating DNA - DNA alone gives this band. We usually don't see it
(we routinely treat our preps with DNAse).
Member, Institute for Cancer Research,
Fox Chase Cancer Center, Philadelphia, PA
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