ChIP and qRT-PCR - (Feb/25/2008 )

Hello everyone!!

I just wanted some expert advice on quantitating ChIP data with qRT-PCR. In brief, I am looking at the upregulation/downregulation of a subset of genes involved in podocyte differentiation by two transcription factors (one is a co-suppressor for the other). I have performed ChIP and I usually have an Input, GAL4 (as a negative control, the cells I use are from a mouse), Ac-H3 (as my positive control) and my two transcription factors. I'm using SYBR Green for RT-PCR and performed an assay last week. My plate set up included:
- A check sample which was an additional Input sample at 100%, 50% and then 10% concentration (all in triplicate)
- My input sample, GAL4 and my two transcription factors (all in triplicate and all from the same ChIP assay) with primers against a site in the Bak promoter which I know my transcription factor binds to
- The same samples as above with the same check samples except primers were directed against a region in the Bak promoter to which my transcription factor doesn't bind.

Does that sound reasonable?

Also, I have two peaks in my melting curve. I'm assuming it's primer dimers and I have tried all kinds of primer design programmes to try and eliminate primer dimerisation.

Can anyone help me, I would be so grateful.

Many thanks

H.

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Hi Hayleyuk,

am a newbie to chip-chip related qPCR as well, that's why I can only comment on last prob:

If you're not sure what second product in melting curve is, just run sample after qPCR on agarose gel and have a look

if you can't get rid of secondary product and it's got a lower melting temp than target product just add melting step before fluorescent detection (I mean an additional step each cycle after annealing/elongation with a temp a bit higher than melting temp of by-product)

hope I could help & good luck

kylvalda

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