use of housekeeping gene in RT PCR chIP - (Jul/25/2010 )
Hi, I'm a technician who is starting to set up my experiments for a ChIP assay. Basically, I have a control and a treated sample, and SYBR green primers for my region of interest from SA biosciences etc. So I have gone through several manuals to look up sample calculations, and it seems like % input is the most common method of reporting data. I will also be calculating using the delta-delta Ct method. However, at what stage does using the housekeeping gene (in my case, I will be using GAPDH) come in? When I have done these calculations previously, I normalized the ChIP DNA fractions to input, checked the % input, then adjusted the delta Ct for normalized background (delta Ct normalized ChIP- delta Ct normalized IgG), then did 2^-deltadelta Ct, then did a second delta delta Ct between the Experimental sample-control and then did another fold change. So when would I use the data for GAPDH? This is my first time setting all of this up, so I'm a bit confused. Any help will be appreciated!
You would use the GAPDH data in lieu of your IgG data. Instead of normalizing your samples to IgG you would do it to GAPDH the same way you are for IgG. In my experience using a control gene tends to be better than IgG because my IgG samples tend to be very noisy or not come out at all. I actually like using something that comes up big in my qPCR that has many copies in the genome such as LINE1 or 28s rRNA.
Hope that helps
Thanks for your response. So do I disregard the IgG data entirely? I should still run the qPCR with the IgG samples, right?
Is it common for the Ct values for GAPDH to be higher than my target region... my Ct values for GAPDH are in their 30s, whereas for my target region, around 26. I'm thinking my initial sample concentration was too dilute..?
Well I use the IgG samples as a qualitative control; i.e, my samples should have lower Ct's than my IgG, but I don't use it to quantify the data. Although, as I'm sure you have seen in the literature, some people do use IgG for quantification; but for me it's too variable and does not always amplify to be of any use.
As for Gapdh having higher Ct's than your sample, well I imagine that is what you would expect for whatever factor your ChIP'ing for? i.e, you don't expect to get much enrichment for Gapdh, right? So of course it's going to be lower. That's why I suggested using a gene that is present in multiple copies in the genome as a potential negative control, so even though your %input is low, it comes out cleaner on your qPCR because there is more of it.
Overall, I think the biggest issue for ChIP in the literature is that there are a number of ways in which the data is reported. Each way has some limitations...such is life in research!