>100% input in histone ChIP qPCR - (May/29/2013 )
i've been having a bit of trouble working out proper conditions for a ChIP qpcr for a transcription factor of interest, but for now, i'll just discuss some other issues that have popped up with histone ChIPs. recently, i performed a qpcr on ChIPs of H3K4me3 and H3K27ac and got some interesting results to say the least... has anyone ever come across a percent input value of over 100%? i'll list the details below:
-250 ul input, 170 ul hist ChIP (i calculated this to be a ~0.68x dilution)
-after DNA purification, I eluted my input and ChIP samples in the same volume. I then nanodropped my input and performed a 9.5x dilution to obtain a DNA concentration in the linear range.
-So, compared to my histone ChIP samples, I calculated the overall dilution of my input to be equal to (0.68)(9.5)=6.46
-Here are my raw Ct values:
-Input - 23.644
-H3K4me3 - 21.126
-H3K27ac - 20.589
-Taking into account my dilution factor (and my primer efficiency), my adjusted input Ct value came to be 21.706.
-My resulting %inputs for my H3K4me3 ChIP and H3K27ac ChIP were ~150% and ~217% respectively.
I realize that %inputs for histone marks are to be much higher than for transcription factors... but this is just insane! has anyone ever come across this before? please let me know. thanks so much in advance
Interesting! Theoretically it doesn’t make sense. The %Input of histone mark is normally high, e.g. %Input of the H3K4Me3 is in the range of 1 to 10. It looks like the PCR efficiency is different from your input and histone ChIP DNAs. Have you tried to do qPCR with diluted histone ChIP DNAs or with your undiluted input DNA? Also have you seen any difference among different regions for H3K4Me3, like promoter vs coding regions?
If you used a column to purify the DNA you might be eluting off different amounts of your initial sample.............this is why you don't generally want to overload a DNA column..........not only do you lose the ability to bind all the DNA, but its much more difficult to elute all the bound DNA off the column