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qPCR on chIP product - (Jul/02/2010 )

Hiiiii
I am new to this technique. I want to perform a qPCR on my chIP samples.What will be my control for comparing my result and how to calculate.

help me...........

-sssss-

sssss on Jul 3 2010, 01:14 AM said:

Hiiiii
I am new to this technique. I want to perform a qPCR on my chIP samples.What will be my control for comparing my result and how to calculate.

help me...........



Hi...first of all you're probably better off posting ChIP questions in the ChIP forum...

Second...I like to have a positive and a negative control for my ChIP...for negative control I use something that shouldn't be bound much by the factor I'm looking at (or has a well known histone modification profile) such as LINE1...for a positive control, you need to search the literature and find a gene that is regulated by the factor your interested in. If your doing ChIP for histones this should be pretty easy to find a good positive control.

As for how to calculate, I typically do %input, there's some old posts in the ChIP section on how to do this I think. Others use fold over IgG, but I find that to be noisy and unreliable, at least for what I do...

MM

-Mighty Mouse-

Mighty Mouse on Jul 5 2010, 06:52 PM said:

sssss on Jul 3 2010, 01:14 AM said:

Hiiiii
I am new to this technique. I want to perform a qPCR on my chIP samples.What will be my control for comparing my result and how to calculate.

help me...........



Hi...first of all you're probably better off posting ChIP questions in the ChIP forum...

Second...I like to have a positive and a negative control for my ChIP...for negative control I use something that shouldn't be bound much by the factor I'm looking at (or has a well known histone modification profile) such as LINE1...for a positive control, you need to search the literature and find a gene that is regulated by the factor your interested in. If your doing ChIP for histones this should be pretty easy to find a good positive control.

As for how to calculate, I typically do %input, there's some old posts in the ChIP section on how to do this I think. Others use fold over IgG, but I find that to be noisy and unreliable, at least for what I do...

MM




Thanks for the reply

Can't we take only IgG or Input sample as control

-sssss-

Thanks for the reply

Can't we take only IgG or Input sample as control




Well if you take just your %input or fold over IgG, how do you know what you're seeing is real? %Input doesn't tell you anything about whether your ChIP actually worked, but if you compare your target to something that should not have been pulled down during ChIP (like an intergenic region, or transposable element or something) then you can have confidence that what you are seeing is real. The reason I would not simply compare just to IgG is that different pre-immune serums that you get from different companies will give you different amplification levels for your IgG; so you could just look around and try and get the lowest amplifying IgG possible so you can say your ChIP worked, but what does that really mean? Although I think, in general, it makes a good qualitative negative control...

MM

-Mighty Mouse-