2 replies to this topic

[Liinda]

I have seen that a lot of people use DEPC-treated PFA for fixing tissue that will be used for in situ hybridisation or will later want to extract RNA but is this really necessary? Surely, given that fixatives cross-link proteins and RNase is a protein, it will be rendered inactive by fixation - this is the whole point of using fixatives isn't it - to render inactive endogenous components that could damage the RNA or protein you are searching for?

Can anyone shed any light on this?

Thanks a lot!

Edited by Liinda, 16 December 2011 - 04:44 AM.

[David C H]

Ditch the DEPC. I've done a lot of RNA work. I stopped depc-treating solutions years ago. We never used DEPC-treated PFA for fixing tissues. I prefer to use milli-Q or ultra-filtered or double-distilled H2O, whichever of those options is available and work carefully from there on out. I used to DEPC-treat glassware, but now I prefer to just rinse with ultra-filtered water. I know some labs that use only the house single-distilled or deionized water for in situ hybs. I change gloves a lot, work clean, have glassware/plasticware that is only used for RNA and I know of other labs which do things similarly. If you are working with RNA a lot, DEPC is just not feasible.

[Liinda]

Thanks for the advice David C H!

Yeah, it really didn't make sense to treat PFA with DEPC!

Although I am surprised to learn that you use ultra-filtered water, I'll stop being so paranoid now.

Thanks a lot!