I am doing ChIP in Arabidopsis to determine Histone modification. My experimental details are:
- Sonicated 300 µl of chromatin. Centrifuged and from supernatant (~270µl) kept 10 microlitres as INPUT. Remaining supernatant was make upto a volume of 3ml using ChIP dilution buffer and equally divided into 3 tubes- No Ab, Ab-1, and Ab-2. After immunoprecipitation, immunoprecipitated DNA and INPUT DNA were precipitated and finally dissolved in 20 µl of sterilized water. INPUT DNA, as well as IP DNA, was 10 fold diluted and 2 µl were used for ChIP PCR and ChIP-qPCR. I have confusion about ChIP qPCR data analysis . How to implement "%input calculation method"? How to determine dilution factor? And after percent input calculation method is it required to normalize to a reference gene like ACTIN7??
It would be really helpful if anyone could provide some tips to calculate ChIP data.
Hi Garima,
I hope the below link will help you understand the calculation part well:
https://www.thermofisher.com/no/en/home/life-science/epigenetics-noncoding-rna-research/chromatin-remodeling/chromatin-immunoprecipitation-chip/chip-analysis.html
The number 6.644 depends on your dilution factor. If your input is 1%, then the dilution factor is 100 fold. If input is 10% then dilution factor is 10 fold. So, you need to first calculate the dilution factor based on the amount of input you have taken aside before adding Ab. Then take log2 of that number and calculate the Adjusted input and proceed with the instructions in the above link. I hope this helps.
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