RT-PCR using formaldehyde cross linked samples - RNA extraction using CHIP crosslinked samples (Nov/15/2007 )

Has any one tried extracting total RNA from formaldehyde cross-linked samples?

I have some formaldehyde crosslinked cell pellete in the -80 freezer. I tried chromatin immunoprecipitation realtime PCR on them and they seem worked.

Now, I wonder if I may resuspend the formaldehyde crosslinked cell pellete in something like the RLT buffer from Qiagen and extract total RNA for realtim-RT-PCR study.

I just want to get the total RNA out of these cells, not worrying about sonication, IP or anything

If any one has done that, or at least extracted RNA from this kind of samples, could you kindly share some tips with me?

Any comments are highly appreciated Thank you!

-Plasmids-

QUOTE (Plasmids @ Nov 15 2007, 07:13 PM)

Thank you!

I've tried it without reversal of crosslinking (like below) with some success. I get maybe 50% yield or less though.

1) Ethanol precipitate out of the crosslinked chromatin (I use glycogen for the precipitation).
2) Redissolve in TE and add RNase inhibitor, bring up to 0.5% SDS and add 20ug prot. K
3) Incubate at 50C for 60min
4) Extract with Trizol or column based kit

If you're going to try reversal of crosslinking at 65C first, I would suggest not doing it at a high pH, since RNA will degrade. At that temp it's probably safer at pH 7 or less (but the lower pH may effect the efficiency of reversal of crosslinking).

-KPDE-