Housekeeping genes for rat brain - (Mar/03/2008 )

Hello,

I'm back again with another basic question! Currently in my PCR (old school with gels, we don't have lovely new-fangled real time malarky) of rat brain samples I am using GAPDH as a housekeeping gene however, I am planning some experiments involving hypoxia and I believe GAPDH expression can be altered in hypoxic conditions. I was wondering if anyone had experience with rat brain PCR and could recommend a suitable alternative, would beta-actin be any good perhaps?

Cheers,

Helen

We use beta actin as the reference housekeeping gene for RT-PCR in rat brain.

Ginger

Thanks, I thought it would be but I get paranoid about these things!

> would beta-actin be any good perhaps?

We did not use mouse brain, but we know that actin can change a lot dependent on your treatment.

We now do is use 3 different Housekeeping gene HPRT, 36B4 (mRNA for ribosomal protein), and beta-actin.
For regular PCR (just take a look on gel), as long as one of them consistent we assume all samples consistent.
By the way, we do quantify bends from agarose gel as our reference.

If you do real-time PCR consistent is a dead serious issue for obtaining a usable data.
There are a long list of reviews discuss such issue, and short conclusion is NO such gene consistently express in every conditions, every tissues and among individual subject.

Best can do is select best one from a pool of genes, and maybe using more than 1 internal control for normailzation.

Good luck