Problems with Housekeeping/Endogenous Control - (Mar/06/2008 )
I've run into a little problem w/ my endo. control. We have been using GAPDH with no problems for the last couple of months (i.e no variation in expression from our control and treatment groups, delta delta ct method). However, in my most recent set of data, I'm seeing up to a ~0.8-1.0 CT difference in GAPDH expression between the treatment and control groups. This would be a huge set back to my project if I had to go back and rerun these samples with a new control gene, but I am unsure as to whether this data is usable. Any ideas would be helpful. Additionally, if anyone knows of a good housekeeping gene for adult rat hippocampus that would be great. Thanks.
Is there any difference in the delta ct of your gene of interest vs GAPDH? Because if the ratio is unchanged, I would simply guess that you had less RNA, so your cDNA concentration was lower than the other experiments you made. I would still suggest to test another house keeping gene, to make sure that GAPDH is still stable.
We use 36B4, a ribosome subunit. Pretty stable!
Hey thanks for the quick reply, my delta ct values are only slightly different ~0.3, which means that you are probably right and I loaded a little less RNA. Does this mean that I should be fine as far as using this data?
Could you tell me a little more about 36B4, I'm starting to get a little scared about using GAPDH. What kind of treatment (manipulations) are you using on your rats. Thanks.
GAPDH is a risky HKG to use, as it has been shown to be affected by several situations, specially anything that will alter cellular metabolism. Also, I would always recommend to use, at least and whenever possible, two HKG, making your results more reliable and less prone to error due to variations in your HKG.
I don't have experience in hippocampus, but in mouse hypothalamus, beta-actin, beta2microglobulin and 18S seem to work well.
I'm a little wary of using actin because our experimental procedure has be reported to induce various forms of actin expression. I plan on sticking with GAPDH for the next couple of runs, and eventual switch over to something less "risky". Thanks.