ChIP - a couple of Q's about real time validation - Too much background? Compare to IgG or Input? (Nov/20/2007 )

Hey hey

Sorry to bother you all again but everyone I need to talk to here at work is away sick...

Ok, so I did a real time PCR to check my ChIP products. I am using GAPDH (+ control) and MYO-D (- control) with H3K9. Looking at the amplification curves it looks like it has worked.
For GAPDH I get a CT of 23 (for H3K9) and 29 (IgG).
For MYO-D I get a CT of 29 (H3K9) and 30 (IgG).

So I'm doing these test real time PCRs to test the quality of my ChiP products before I do the chip-on-chip. My questions are:

* is the signal for IgG too high (therefore too much background?) - I used 2x10^7 cells per IP, purified with a kit, eluted in 50ul and used 1ul in PCR.
* to calculate the fold enrichment do I compare H3 to IgG or H3 to input or H3-IgG to input?
* what stats do you use to determine if your enrichment is statistically significant enough to proceed with chip-on-chip?

I hope someone can point me in the right direction, I have been reading papers for days now and can't seem to find any definitive answers. Perhaps I'm just looking in the wrong place!

Thanks in advance

Clare

Hey hey

Sorry to bother you all again but everyone I need to talk to here at work is away sick...

Ok, so I did a real time PCR to check my ChIP products. I am using GAPDH (+ control) and MYO-D (- control) with H3K9. Looking at the amplification curves it looks like it has worked.
For GAPDH I get a CT of 23 (for H3K9) and 29 (IgG).
For MYO-D I get a CT of 29 (H3K9) and 30 (IgG).

So I'm doing these test real time PCRs to test the quality of my ChiP products before I do the chip-on-chip. My questions are:

* is the signal for IgG too high (therefore too much background?) - I used 2x10^7 cells per IP, purified with a kit, eluted in 50ul and used 1ul in PCR.
* to calculate the fold enrichment do I compare H3 to IgG or H3 to input or H3-IgG to input?
* what stats do you use to determine if your enrichment is statistically significant enough to proceed with chip-on-chip?

I hope someone can point me in the right direction, I have been reading papers for days now and can't seem to find any definitive answers. Perhaps I'm just looking in the wrong place!

Thanks in advance

Clare