I try to do qPCR for dark-operative protochlorophyllide reductase (DPOR) subunits (chlL, chlB, chlN) in Norway spruce (Picea abies Karst.). I have the specific primers for these subunits and also the primers for 23S, because DPOR subunits are plastid encoded. But there is a very quick and very high transcription when observed through qPCR curves (after 5 or 6 cycles already) and very fast it is saturated. On the other hand, my other primers (for DPOR subunits), there starts the transcription after 20-25 cycles, so it is not comparable with 23S. So what can you recommend me? Which another, constitutively transcribed gene should I use? Thanks.
I only work with human samples, so I'm not sure how well this will translate to plants, but, our lab uses GAPDH as an internal control, and that seems to be pretty common, although there is some question as to how consistent it is. (I also think it's highly conserved, so it might work in plants as well). I've also used b-Actin and tubulin as normalizers on occasion.
Although it's done with human samples, you can take a look at this paper, and might be able to use a homologous gene:
http://www.gene-quantification.de/radonic-hkg-2004.pdf
You can try actin, GAPDH as mentioned above, EF1, ubiquitin, tubulin. On general they are working quite well with several plant species. Good luck!
You should test at least 2 different genes over the samples and check the most stable for the samples like hTERT, RNAse P, GAPDH, B2M, and the expression should not be very different from your gene of interest. Good luck!!!
It is indeed better with 2 different genes, some people also use rRNAs, or ribosomal protein genes
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