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Old 02-04-2009, 03:01 AM   #1
Danny
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Question [Sample prep] Enrichment and aspecific control use with qPCR

Hello,

I am interest in doing a ChIP for ChIP-seq. As a control I have seen that DNA concentrations with qPCR and specific primers are determined. From these concentrations is an enrichment value calculated. Before the enrichment value is calculated, many times people correct the concentration of the samples for the input concentration. But what I also see is that some people are using a control for aspecific binding (Rabbit IgG control). But is this a background sample, because with background I think of an addition to my sample value like in spectrometry (and in your sample is no IgG antibody present only the target antibody).
So results this in concentration target antibody sample - concentration IgG sample?
or in concentration target antibody sample/ concentration IgG sample?
And how good is IgG as a control? Is it really necessary to use an a specific binder? And what does it say about mine ChIP? Can you use it as a ChIP to ChIP variation value (the difference between the enrichment's).

A lot of questions, I am really curious to your opinions.
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Old 02-04-2009, 03:51 AM   #2
Chipper
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Hi,

for qPCR I would say it is better to use a non-specific region where your ChIP is not supposed to be enriched as a background control. You use the input (ideally a dilution series) as a reference since each primer pairs will give different amplification curves.
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Old 02-04-2009, 04:36 AM   #3
Danny
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Quote:
Originally Posted by Chipper View Post
Hi,

for qPCR I would say it is better to use a non-specific region where your ChIP is not supposed to be enriched as a background control. You use the input (ideally a dilution series) as a reference since each primer pairs will give different amplification curves.
hi, thanks for your reply. But why is most of the time also IgG taken as control? Is it than more a control for the Solexa as kind of background of the sequences you pickup?
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