Jump to content

  • Log in with Facebook Log in with Twitter Log In with Google      Sign In   
  • Create Account

gubi

Member Since 17 Dec 2012
Offline Last Active Jan 29 2013 06:19 AM
-----

Topics I've Started

RNase inactivation during IHC prior to LCM

17 December 2012 - 06:11 AM

Hi!
I am currently working to optimize a protocol for detection of specific cells in the intestine aiming at a strong specific signal and a high RNA quality. After IHC the cells must be captured individually using LCM and then analyzed for gene expression levels. I work with fresh-frozen tissue, stored at -80.
The RNA quality is initially good (not perfect but OK), but after (cryo) cutting, fixation, immunostaining and contrastaining the quality is very poor. I have optimized the IHC protocol to be quite fast (only around 20 min in aquous solutions), and I have added enzymatic RNase inhibitors to all aquous steps. As this doesn't seem to be enough, I am currently looking for efficient chemical RNase inhibitors. I believe (at least part of) the problem is with endogene RNase's, so the inhibitor must be able to penetrate the tissue (8µm thick). I have been recommended n-ethyl maleimide (NEM), but have not been able to find any information about how to use this.
So: Any good advice in general?
Any good ideas about chemical RNase inhibitors?
Any ideas about how to use NEM as an RNase inhibitor?  
Thanks!

Home - About - Terms of Service - Privacy - Contact Us

©1999-2012 Protocol Online, All rights reserved.