[Histonet] what do people for ISH

[Mikael Niku]

Hello Jennifer, Emily, and others,

I think it's really best to be as careful as possible when one is 
starting RNA work. But if the lab has been working succesfully for 
years, I don't see why they should change the practices. Only if they 
are now looking at very rare RNAs, requiring top quality materials for 
maximal sensitivity, this might be relevant.

DEPC treatment of solutions is in my experience unnecessary, if one uses 
high quality materials. So one can do without this highly toxic stuff.
MilliQ water is as such RNAse free (I tested ours by incubating my RNA 
probes a few days at room temp... no detectable degradation). Using 
RNAse free glassware & tools, RNAse free chemicals, and of course 
gloves, the solutions will be RNAse free without DEPC.

The endogenous RNAses are probably really the most serious threat to 
RNA. Tissues like pancreas are a nightmare, due to high enzyme content. 
So after killing the animal, you need to work quickly and use effective 
RNAse inhibitors / inactivators. We don't have separate processors, 
microtomes, or cryotomes for RNA work, and I'm fairly satisfied with the 
results.

With best regards, Mikael