I'm doing a crosslink RNA IP. Briefly, my cells are crosslinked with formaldehyde prior to cell lysis and immunoprecipitation. After eluting my sample and reversing the crosslink, RNA is extracted via TriZol and ethanol precipitated. The RNA is then reverse transcribed to cDNA, and subjected to real time qPCR.
I've read a few protocols (most for ChIP, some for RIP) recommending sonicating the lysate after IP. Is this necessary when the downstream analysis is RT-qPCR? Any input would be appreciated. Thanks!
Hello,
according to experience in our lab, I would rather sonicate cells. It also depends on whether your protein is nuclear or not. In case of nuclear protein, the lysis of crosslinked cells may not be complete and therefore you will lose part of your sample. We have experienced problems with worse lysis of formaldehyde-crosslinked cells. So, I would not skip sonication.
good luck!
I agree with Pavlina, you have to sonicate the samples BEFORE IP (after crosslinking). But are you talking about AFTER IP? I don't know what should be the reason for sonication after the IP...
Thanks for you reply! My concern was that without sonicating, crosslinked protein-RNA might fall into the interphase layer during TriZol extraction for RNA, preventing complete RNA isolation. It seems that reversing the crosslink by heat treatment is needed and not sonication. Thanks again for the replies!
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Biomolecules
- Nucleic Acids
- RNA