Quantify DNA before ChIP PCR (View forum version)



Mindbomb

Posted 23 August 2012 - 04:30 AM

I did a ChIP experiment for a transcription factor. Just after the chromatin shearing I separated 5% as input. Now as everything is finished I would do the QPCR. I'd like to find out the % from input. Do I need to quantify the ChIP and input samples before PCR and standardize them to the same level? Thanks!

chabraha

Posted 27 August 2012 - 09:10 AM

Short awnser is no.

Mindbomb

Posted 31 August 2012 - 08:34 AM

Thanks! I would like to compare protein binding to DNA between two samples. Is the best way to just use the formula 2^(Ct background-Ct binding site) in order to find the fold change, in which case the Ct background is measured based on the same IP sample? I was worried about the DNA quantity as differences there can lead to different Ct values between samples. Is it necessary to incorporate the input sample somehow in the formula?

chabraha

Posted 03 September 2012 - 09:49 AM

the delta-delta method is fine for ChIP............the way i do it is by calculating a %Input value for each target by using the input Ct value...........SA Biosciences has a good description on the math involved for this step. Basically you will have a % Input value that tells you how much of the total avaliable target region you were able to IP..........I calculate a %Input for both a specific IP (TF of interest) and a non-specific IP (IgG or isotype) I subtract the non-specific from the specific and now you have an IgG corrected %Input. Look at this value at a region known to bind your TF of intererst and a region known NOT to bind your TF of interest..............the differences there should be significant in order to call it a successful ChIP.