housekeeping genes - (May/04/2007 )
Hello, I'm new in this but been reading for a while and learn a lot.
I was wondering (following on previous discussions) what would be a good housekeeping gene, or internal control for RT-PCR. I'm thinking the usual suspects: GAPDH, B2M, TUB and 18S, but any other suggestions will be more than welcome.
Ah!, and this is in human, a whole new subject for me mousey person. though I assume and hope PCR technique doesn't really change, just the sequence. a shame i can't use my thesis primers...
I think it very much depends on the treatments you use. First you have to make sure that the housekeeping does not change in your experiments.
What I usually do is to try a few possible ones (do a literature search beforehand to see if they are likely to change or not) along with 18S and compare them, seewhich one is the better. In my current lab they routinely use 18S, but other colleaques said that it is not that good because it does not reflect the amount of protein mRNAs well. On the other hand, I read a paper comparing different housekeeping ones and they concluded that 18S is the most reliable one.
So, test a few of them.
We usually dilute cDNA for 18S well, to make it more reliable. We also use Hgprt1 as a housekeeping. It was very convinving when both Gapdh and Hgprt1 gave exactly the same result with taqman (~90%).
I think Qiagen has a got a nice booklet on real-time pcr and they have a few good hints with respect to housekeeping. Let me know if you`re interested in that and I`ll try and find it for you.