Removing RNA from DNA during ChIP Assay - (Jul/23/2008 )

I have been having a problem for some time and I hope someone will be able to give me some advice!

I am doing ChIP assays on yeast cells. Following the IP step, our lab reverses the formaldehyde crosslinks (65C, usually overnight) then treats the samples with RNase and proteinase K. Following this step we do 2-3 phenol/chloroform extractions, then an ethanol precipitation. My problem is that I am getting very small digested RNA fragments with my purified DNA (<100bp, although probably in general > than 40-50bp). When I do the subsequent PCR steps, I sometimes (unreliably) get non-specific bands that are about the size of these small RNA fragments, so presumably my primers are amplifying some complementary RNA sequences from this leftover stuff. I have tried many things to optimize the PCR to avoid this problem (changing the DNA concentration, primer concentration, buffer, titrating the MgCl2 concentration, changing the annealing temp, etc.) but to no avail. The reason this concerns me is that when this small product shows up unexpectedly, it affects the amplification of my target bands. Since I have spent a long time trying to get rid of this at the PCR step and have yet to figure out what causes this product to show up, I thought I'd try to get rid of the RNA itself.

We generally add 33ul RNase A (at 10mg/ml) to 1 ml TE and then add 400ul of this solution to our samples (after the crosslinks have been reversed) and incubate at 37C before the proteinase K step. I have tried increasing the amount of RNase used, as well as using an RNase A/T1 cocktail from Ambion (described here:http://www.ambion.com/catalog/CatNum.php?AM2288. They also talk about why this is better to use than RNase A alone, which supposedly can give you small RNA fragments that co-purify with your DNA, as I have been seeing). However, neither of these solutions seem to get rid of this small RNA. I have compared the treated samples to RNase untreated samples, and the RNase is DEFINATELY working, it's just leaving the small stuff behind (what it can't digest). I have also tried treating the already RNase treated samples with DNase to make sure that what I am seeing is not DNA, and it does not appear to be (the sonicated smear of DNA is gone after DNase treatment, but this small stuff is still there). I am wondering if there is a better DNA precipitation method than what we use (200mM NaCl, 2 volumes cold 100% EtOH and ppt at -20C for 1 hour or longer - I usually go overnight at least) that will selectively leave behind these small fragments? Alternatively, does anyone have any tips for during the P/C/I extraction steps that I should keep in mind? I do my best to not disturb the interface, leaving behind some sample to do so, and can definitely see that the interface gets much more clean with each extraction.

I am probably just going to switch to doing the DNA purification using QIAgen PCR purification columns, but wasn't sure if I will get as much DNA out of this method.

Thanks for any tips!!

(wannabe) lab ninja

Hi there,

Correct me if I'm wrong, but I think you should be adding your RNAseA to your NaCl ON at 65deg. Then add your PK at a lower temperature.
Clare

Hmm.. not sure about that.

We do our RNAse treatment at 37 for 30 min and it seems to work fine.

As for the P/C extraction, i've heard that the pH of the phenol changes the solubility of the nucleic acids. If your phenol is acidic, the RNA and the DNA will be kept in the aquous phase. If you have a phenol at pH 8, only the DNA should remain in the aquous phase. You may want to check the pH of the phenol you are using.