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





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