Problem with qPCR of ChIP input - (Jun/04/2008 )

Hi everyone,

I'm new around here, as well as new to ChIP... I have two questions, one general and one more specific, and would greatly appreciate help/comments!

1) Why are agarose/sepharose beads usually used for ChIP? I know that Dynal magnetic beads are also used, but is there some reason for a specific choice of material for the ChIPing beads? Reason I'm asking is because for my specific application I want to use polystyrene-ProtA beads, and I'm just wondering if the bead material might hurt the ChIP?

2) I have been doing ChIP on mouse cells with the anti-H27Me3 Ab, and then putting the samples first through a multiplex normal PCR to amplify (20 cycles), then through SYBR green qPCR to quantify. My IPs look fine, with Cts around 25-30, and my IgG control is slightly on the low side bust still ok, but the weird this is, I seem to be getting almost NO DNA in my INPUT sample. Those Cts, if I even get any, are around 36-38.
The only difference in my IP and input sample treatment is that I wash the IP beads, but I don't wash the input beads.

Any clues? I'm desperate, because this is a very repeatable phenomenon (I wish my other results were so repeatable...) so there must be some systematic error!

Thanks much much much!

Angela

The material that the beads are made of should make no difference to the experiment. I'm guessing that agarose beads have been around longer than the magnetic beads. The advantage of the magnetic beads is easier use during washing steps, which makes them look pretty appealing. Personally I have had more luck with agarose beads, but thats not to say magnetic doesn't work.

Hi Dave,

Thanks for responding! I am using a unique protocol because I am trying to semi-automate the ChIP process onto a microfluidic platform, so by that nature, what I do is I also incubate the input sample with the IgG beads like I do with the IgG control sample, but after incubation, I do a wash on the IP and IgG control so that only the bound DNA is left, whereas with the Input sample I just keep everything and run qPCR on that. Is there anything in there that you see that might mess things up?

Thanks,

Angela

Perhaps the beads in your input sample are causing the problem. I have never heard of anyone adding beads to their input samples - do you *have* to do this?



[quote name='chipnewbie' date='Jun 5 2008, 02:27 AM' post='138432']
Hi Dave,

Thanks for responding! I am using a unique protocol because I am trying to semi-automate the ChIP process onto a microfluidic platform, so by that nature, what I do is I also incubate the input sample with the IgG beads like I do with the IgG control sample, but after incubation, I do a wash on the IP and IgG control so that only the bound DNA is left, whereas with the Input sample I just keep everything and run qPCR on that. Is there anything in there that you see that might mess things up?

Thanks,

Angela

I think you should still take an aliquot of your sonicated extracts for your input. Adding beads is definitely not a good idea

Semi-automated! Sounds interesting. I'm not sure which aspect you are trying to automate but possibly this reference might be of interest: Flanagin et al., 2008. Nucleic Acids Research, Vol. 36, No. 3 e17

If you can avoid the washing steps with your automation, avoid adding the beads with your automation. If they are agarose beads, it may be casuing PCR problems if there is any carry-over of the agarose. At the 95 degree stage of the PCR, the agarose would probably melt and cause all sorts of problems when it is cooled again to annealing temp and even at 72 it could stilll be a solid.

Hi!

Sorry I've been MIA - I had commencement!

So I took your advice and took out input material without beads, but it made no difference in the PCR! I still get IP>Input!!! I am at a loss as to what to do. The protocol was working fine before, and so I've tried reverting to my old, supposedly non-optimized protocol to see if that will fix things... But I will know some time later this week after I process everything.

Basically, my automation is done using a microfluidic chip, where I load the cells into this plastic chip, and then all the lysing and everything happens on the chip. However, this means I can't sonicate the DNA, so I use the Micrococcal Nuclease digestion method instead...

Anyways, if anyone has ANY other ideas about this at all, I'd be grateful and happy to try out anything!

Thanks again for your inputs!

Angela

Thanks for the reference!