I don't find housekeeping genes: what should I do? - (Sep/26/2013 )
I am trying to normalize my experiment using SYBR Green qRT-PCR kit for RotorGene. I used 8 endougenous controls (b-actin, GAPDH, HSP90, PGK1, b2M,18SrRNA, HPRT and GUSB), however all of them varied between the control and treated samples. What should I do?
Most of the housekeeping genes should not be affected by any treatment. Do they vary in a consistent way among samples? If they vary wildly, your results should not be trusted.
The Ct was higher than 0.5 between control (untreated) and treated. I don't know what I do. The 8 housekeeping genes that I tried are the most used in qRT-PCR, not? Do I have that to try other endogenous controls? Or is there another way?
>>The Ct was higher than 0.5 between control (untreated) and treated.
That does not tell you that your housekeeping genes vary. They could have different Ct values between control and treated samples, resulted from unequal loading of RNA, but they should have a similar pattern.
If you want to try other ones you could also try Lamin B1 and RPLP0, they work fine for me...
A difference of 0.5 doesn't look quite significant
Thanks all for the help! I was not paying attention to loading of cDNA between the groups. Now my experiment works. Thanks a lot!