Yield after ChIP Assay - (Nov/07/2008 )

I've done some ChIP experiment and got very low enrichment I guess

But since I'm new to this technique, I am curious if it's normal or something's wrong, so I want to share it here and see if you guys have any thoughts

Tissue: Mouse Liver, 100ug DNA to start with

Target: acH3, H3K4me3, H3K27me3 (Ab from Upstate or Abcam), also include an IgG as negative control

Quantification: With realtime PCR, self-designed primers and SYBR green, total of 50 cycles


So after ChIP, I resuspend my pellet with 40ul and use 0.5ul to do realtime on a ABI-7900 384-well plate setup, final reaction volume of 12ul

The INPUT come up at around 25-30 cycles, and some of my samples come up at around 45, some never come up, and the IgG is usually even later than my samples.

When I calculate the enrichment (ratio of sample / input), I got like at most 0.2%, and in some cases 0.00x%, is it possible?

The reason I think they might not be crap is because the CT is still smaller than IgG

But I wonder if it's normal to get enrichment of that range.....

Hi there,

If your samples are coming up at about 45 cycles I would try and concentrate your ChIP DNA and repeat the PCR.

Clare

Hi there,

If your samples are coming up at about 45 cycles I would try and concentrate your ChIP DNA and repeat the PCR.

Clare

Thanks Clare,

although I don't quite get it...

why if the sample comes up at 45 cycles you would suggest concentrate the DNA?
cos it might be equally probable that the region of concern has no binding to the histone, so the enrichment is low and so the CT is large
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That's true With our qPCR we always find that if the CT is quite a high cycle number we get an ugly spread of our replicates. That may just be us though

I agree with Clare - you should probably try to either resuspend your IP'ed material in a smaller volume, or use a larger volume per reaction (0.5ul sounds like a nightmare to accurately pipette in the first place...). Cts that high essentially mean you're looking at something that is present in a single copy or two in your well, so you're getting close to a 50:50 chance of having some material in there to start with...

Do you have a positive control ab? And did you check your primer efficiency? - the lower the efficiency, the more error you will introduce when working with high Cts.