4 replies to this topic

[gubi]

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!

[Curtis]

After fixation and permeabilization anything can penetrate the tissue.

[Myworkismyplay]

I would suggest trying to do everything on ice if you are not already. What is your RNA extraction method post-IHC?

Edited by Diana Albarado, 26 December 2012 - 08:10 PM.

[gubi]

Curtis, on 23 December 2012 - 11:03 PM, said:

After fixation and permeabilization anything can penetrate the tissue.

I would have thought so too, but even the addition of 3U RNAsin/µl ab dilution ect. does not seem to preserve my RNA. So I believe I have a lot of endogene RNases that just does not get inhibited fast enough:-(

[gubi]

Myworkismyplay, on 26 December 2012 - 08:06 PM, said:

I would suggest trying to do everything on ice if you are not already. What is your RNA extraction method post-IHC?

Thank you for your suggestions! I have performed the IHC on ice (or actually in the fridge w. ice cold reagents), and it doesn't help. I purify my RNA with a column kit (Arcturus PicoPure) that I know works well, and always include a control which is fine.