Strategy for finding RT-PCR housekeeping gene
ucgatan at ucl.ac.uk
Tue Apr 27 10:32:08 EST 2004
On Mon, 26 Apr 2004, Paul K. Farmer wrote:
> We're performing RT-PCR on RNA extracted from cultured cells treated
> with several potent reagents (alone and in combination with one
> another). Our gene-specific primers work great (good specificity,
> efficiency, reproducibility, etc).
> Unfortunately, our treatment effects are modest two-fold changes.
That's one-fold good enough!
> Furthermore, we have not been able to identify any primers corresponding
> to a "housekeeping gene" that provides constant mRNA levels in response
> to cell treatments. We've tried several primer pairs used by others in
> different systems, but the abundance of these transcripts have also
> varied with cell treatments.
Perhaps your treatments are having some effect on overall transcription
levels, eg killing the cells or making them sick. Have you tried the
classic housekeeping genes, like GAPDH, actin and tubulin? If you're
seeing similar variation in all of those, then this would seem likely.
In that case, you might simply use the levels of the housekeeping genes to
normalise the levels of the other genes. However, you do have to worry
about the nonspecificity of the treatments.
Tom Anderson, MRC Laboratory for Molecular Cell Biology, UCL
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