I have a basic query regarding the absolute necessity of internal control genes in ChIP-QPCR experiments. When we work on a particular TF, we are not sure about its binding statistics over entire genome. So how can we decide upon it? Literature won't serve the purpose as binding of TFs is a conditional decision. So exactly where does it serve the purpose towards the accuracy of the results? If we omit it from our analysis, how would it affect the accuracy of the results results and what precautions can one take to avoid any such effects? I mean if I don't analyze any internal control gene (so called), can I still prove my results accurate by adopting certain other precautions? My question is definitely more towards the reviewers as they only can decide over the publication authenticity of such data. If they receive a manuscript with ChIP-QPCR data analyzed without considering any internal control gene, how would they react and what suggestions that have in store? All suggestions are welcome.
We also do so. But we are required to use the same process using IC gene primers in replacement for GOI in parallel. So as to show no change over IC locus as opposed to the GOI locus, reflected by input/ ChIP DNA QPCR ratio.
The less that is known about the genomic occupancy of your factor, the higher the bar for convincing reviewers. There are many controls you should consider including to demonstrate the specificity and strength of binding in your ChIP-qPCR experiment.
1. Non-specific IgG control IP.
2. Non-specific genomic binding sites. It is helpful to include both sites at genes that you do not expect to be bound, as well as sites adjacent to your positive binding sites (this allows you to look at local enrichment, and distinguish between a broad region of genomic association vs. a specific binding site that is more representative of how TFs interact with the genome (picture a discrete ChIP-seq peak in a genome browser track).
3. Negative control ChIP-qPCR on cells where your TF of interest is knocked-out, knocked-down, or not expressed.
What are the controls that you've done?
What if we show Input/ anti-TF ChIP ratio with Input/ NoAb ChIP ratio as a control for each sample under every condition tested? Please suggest.
What is "Input/ NoAb ChIP?" Do you mean a beads only control? That won't do. You need to show that the ChIP-qPCR enrichments at that site are dependent on the binding of your factor. Think about the other potential sources of non-specific signal here: your GOI-directed antibody might cross-react with some other TF that is bound at your site of interest, or for some reason, this genomic site may be sticky, and come down non-specifically in the IP with any IgG. A beads-only (no IgG) condition controls for neither of these scenarios.
It is easy to do the non-specific IgG control IP, and to design primers to interrogate genomic loci adjacent to your sites of interest. I would strongly urge you to include these negative controls.
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