qPCR after ChIP - lack of reproducibility - (Oct/12/2005 )
Well. something really strange is happening to my IP samples. When I perform the qPCR to amplify the region I want, it works in the input samples and in all the points of the standard curve, and it is negative in my negative controls. The problem is that in the IP samples I have not consistent result in my triplicates. I mean, in the same same with the same mix some are negative and others are positive. I am sure it's not a pipeting thing.
Have you ever have the same problem? What can I do?
how are you isolating your DNA? You may have residual phenol or ethanol in your DNA sample which could be inhibiting the PCR reaction.
I'm doing phenol:cloroform extraction and 70%ethanol wash, as usual. I prepare a mix for all the reactions, and a sub-mix for each triplicate. If any of them worked I think it could be the problem, but it's working in one or two reaction within the same sample but not in the others.
what machine do you use?
we had a similar problem with the ABI 7700 last spring
it turned out to be a product of two problems combined:
1. there was a minute bit of someone's skin oil on a part of the lamp, causing inconsistent reading in part of the plate
2. there was some residual fluoresence in many of the wells. there was a function of the software where you could do a blank read and see this, but the machine was used by tons of people and no one was taking care of it. the fluorescence had to be very carefully cleaned out. now the machine is checked about once a week, and there are always a few wells that would give you a positive no matter what your sample did. over time this few wells becomes many and your reads are a big old mess. the early symptoms were a small but noticable loss of consistency in your triplicates ... high std dev, one sample that had very skewed results, that sort of thing... ultimately it lead to the what you describe
hopefully this can help you
I have actually had this happen with the ABI 7700 also, wehre individual sensors were dirty and not reading correctly. That could be a possibility, but IMO I think you have some PCR inhibitors, make sure your pellet is completely dried before resuspending the DNA after your final wash. I addition, you could do a second straight choloform extraction to remove the residual phenol after your phenol/chloroform step.
well, thak you guys. I have the MJ Research PC200. I'll look if there is a way of checking the way it is reading. I'll be sure that my DNA is really clean. Anyway, I keep on wondering why IP DNA is doing different that the no antibody or the input DNA. I'm doing again my chip. I'll let you know if I find an answer.
You are at a much lower copy number in your ChIP and that may have something to do with it. Are you treating your input DNA similarly?
Probably it has something to do, but in my standard curve (made with one of the input samples) I have one point of similar amount and it works nicely.
Are you treating your input that same; are you phenol/chloroform, etOH ppt.? It is common when you have low levels of inhibition to have some wells work and some not work. I really think that is what is going on here.
I'm treating all the samples in the same way.