Best house keeping gene for RT-PCR - (Jul/22/2008 )
Generally GAPDH, Beta actin, tubulin, RPL etc are used as a control in PCR, westerns and other assays. But many claim that even these gene expression could vary with cell types. Therefore which gene would you consider best suitable for using as control. In my hands RPL gives best results. It could be completely context Dependant(I compare cancer Vs normal). I wanted to know which genes work good in your experiments.
sorry Calvin, may I tag along your post and ask...
can the same housekeeping gene be used as a control for genomic DNA and cDNA (ie. GAPDH)?
I don't work with housekeeping genes, but I've learned at a conference that it is better to use as many housekeeping genes as possible and that there is no "standard" housekeeping gene, which you can use for every cell line / cell type.
Agree,
Just say for example, I used to work in apoptosis, and someone told me in some cases, b-actin can change during apoptosis, so I used tubulin for western blot housekeeping gene.
Now, I am working with siRNA and I notice GAPDH mRNA level may change as well when different genes were knock-down (compared to 18S)
I guess there's no simple answer, and you have to know your system
Hallo, I succesfully used Bactin in my QPCR experiments, Actually it is impossible for any gene keep the same expresion level in different conditions cell types or onthogenetical stage, but Bact GADPH .... seems to keep expresion level best of all. In QPCR there is statistical method using endogene control (housekeeping gene) and calibrator(sample wihout treatment) and the treated sample to obtain the best results and decrease effect of different house keeping gene expresion
There is no "the best" gene.
In my hands, it was GAPDH.
I normalize for HPRT and YWHAZ, sometimes I add UBC, but with this gene you first have to make sure that it is stable and that you don't have proteasomal degradation.