3 replies to this topic

[Mindbomb]

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]

Short awnser is no.

[Mindbomb]

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]

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.