I have been trying to do cRT-PCR on mRNA extracts to determine a cut site, but my problem is that each time I find myself with digested RNA.
I keep it on ice, use RNaseZap™ RNase Decontamination Solution on all my instruments, and use filter tips. I use a very good protocol for RNA extraction already tested in our lab using phenol/chloroform, that works just fine, but I still find myself with extremely degraded mRNA. I can't use AIA (AurIntricarboxylic Acid), because it blocks any enzymatic reaction downstream.
Does anyone have a recommendation of a solution or a step to add in the protocol to have a better RNA extraction?
You can try turning your mRNA into cDNA with either a 1-step or 2-step RT PCR kit. cDNA is double-stranded and more stable than mRNA.
You're doing everything right as far as keeping your samples on ice, keeping your workstation clean, etc.
Hello
RNA is stable about 2 or 3 months in -30. For long storage, its better to decrease temperature to -80.
Beside this, for better RNA yield, use RNase free microtubes. it enhances RNA integrity. remember, AUTOCLAVING DOES NOT INACTIVATES RNASE.
Katie A S Burnette do you know a good one to recommend me please? I would be grateful, I've been using the superscript IV, but I do many treatments before (RppH, ligation ect...)
Sounds like one of your buffers is contaminated with RNAse. I'd throw out everything and make new with molecular biology grade (18 megaOhm) water. Good water is essential to RNA work. If you have a water polisher (nanopure or MilliQ or what have you) be certain it is being properly maintained. Otherwise just purchase molecular biology grade water.
If possible for your application, storing RNA in TE rather than plain water can help discourage it from degrading from self-cleavage.
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