use of housekeeping gene in RT PCR chIP
Posted 25 July 2010 - 08:37 AM
Posted 25 July 2010 - 08:25 PM
Hope that helps
Posted 26 July 2010 - 07:22 AM
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..?
Posted 26 July 2010 - 06:41 PM
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!