I am planning to test 18s rRNA among a number of putative housekeeping/reference/endogenous genes for real-time pcr for my cells. Now I read that "due to its extremely high expression in most cell types, it can sometimes be challenging to use 18S rRNA as an endogenous normalizer".
My question would be what exactly is the problem and how to best deal with it?
Thanks in advanced.
Part of the problem is abundance, so with a cDNA quantity (per well) sufficient to put most mRNAs in the 20-30 Cq range, 18S will give Cq values of 9-12, thus (for example) any inhibitors in your PCR mix will barely affect 18S signal but may significantly affect test genes, as they'll have twice as many cycles to exert their effect.
If you dilute your cDNA for your 18S samples, then you've just introduced another possible source of error (pipetting, dilution), so that's less than ideal.
The main problem though, in my experience, is that 18S simply isn't a very reliable reference gene.
Ribosomal RNAs comprise anything from 80-95% of the total RNA in a sample, so when you spec your RNA in preparation for cDNA synthesis, you're mostly measuring rRNA. Actual mRNA content could vary wildly without ever significantly affecting the measured total RNA concentration, because so much of it is just...ribosomes.
qPCR for 18S acts more or less as simply a confirmation that your spectrophotometer is still accurate. :-/
rRNAs are subject to different decay pathways, too, so will not degrade at the same rate as your mRNA population, introducing another possible source of error. Generally speaking, ideal reference genes should be mRNAs with expression levels comparable to your genes of interest.
Still, your mileage may vary (depending on cell type/tissue type), so try it out. If your 18S signal varies very nicely in line with your other two reference genes, then it's probably ok.
Thanks for the input, much appreciated.
Quick follow up question: To dilute or not to dilute (the cDNA differently for 18S compared to other ref. genes)?
Mortiz, yes, since there is about 260,000 X more 18S than most other 'qPCR targets' it is highly helpful to pre-dilute the samples at least 1:500 to 1:1000 X more than you would dilute the samples prior to their use in analysis for most other qPCR targets...
I agree with all the suggestions , Actually, choosing a righ reference genes is really difficult and complicated sometimes . I have tried many differnt genes as control including 18S rRNA and rRNA actually is a good reference gene because it is easy to detect and stable. But , the problem I had for rRNA is its abundance. It is too high, so what I suggested is depending on your target gene expression level, if your target gene expression level is low, I don't recommend you use rRNA. And the other reference genes like actin, GAPDH, HPRT, you should test and choose them depending one your sample (cell type, tissue type, or whether your project related with metabolism....). Good luck with your PCR.
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