total RNA isolation from matrigel coated plate - (Nov/08/2005 )

hi,
I need help
I wanted to isolate total RNA from matrigel coated plate but I could not isolate.
I use trizol for isolation. I isolated RNA from collagen, fibronectin and laminin coated plates succesfully.

-dilay-

QUOTE (dilay @ Nov 8 2005, 05:56 AM)

I'm bringing this topic back to the top - anyone have any ideas? I now thinking that matrigel is the reason my RNA is not making it through my purification columns, and also the reason for weird interphase layer issues (posted a few days ago).

Thanks!

-soluene-

Ok, I'll put this here for anyone else who may have this problem:

Do a Proteinase K digest of the RNA pellet. Don't do it in the aqueous phase of Trizol because something in there (phenol?) seems to inhibit PK activity. But if you resuspend the RNA pellet in buffer RLT from Qiagen and proceed with the Proteinase K digest, the matrigel dissolves the the RNA goes happily through the column!

Other options are Dispase (BD Cell Recovery Solution - I think they are the same thing) or MatriSperse digests.

-soluene-

FYI the aqueous phase of trizol is high conc guanidine salts, that's why it inhibits PK

Bizarrely though the RLT is practically the same stuff, so there may be other contributing factors such as residual phenol

-John Buckels-

Hi John-

The Qiagen spec sheet says that their PK is active in up to 800 mM GTC and 3 M GuHCl.. But maybe the concentrations in Trizol are higher than that? That was why I thought it might be the phenol. But in any case, at least I know what to do now!

Thanks for the comment...

-soluene-

As far as I know, RLT contains higher conc guanidine than both those you stated

However in our lab Proteinase K works just fine in 8M GuHCl............

One of those things I guess

But yes, you know what to do now!!

-John Buckels-