Your low mRNA yield is almost certainly caused by Trizol. We have found it does not isolate much (if any) mRNA from young Arabidopsis seedlings, but it isolated plenty of rRNA. The problem gets less severe as plants age and by 11-14 days it's back to normal. Why ... we have no idea - someone suggested at one time the high starch content of young seedlings might be affecting Trizol? We included a comparison of Trizol with a phenol:chloroform/LiCl extraction method in a paper from last year. See Fig. 1 of Box et al. (http://www.plantmethods.com/content/7/1/7) ... the figure caption says it all: "TRIZOL is not suitable for extracting RNA from very young tissues".
I will send you the phenol:chloroform/LiCl bench method we use (and of course to anyone else who asks), but bear in mind it has a LiCl precipitation step at the end so it would lose some of the smaller RNAs you want to keep. You could simply drop that step out. This is what the Box et al. paper did and the RNA extracted in this way was still fine.
T: +61 7 3346 2021
E: j.mylne from imb.uq.edu.au
From: arab-gen-bounces from oat.bio.indiana.edu [mailto:arab-gen-bounces from oat.bio.indiana.edu] On Behalf Of Slotkin, R. Keith
Sent: Monday, 20 February 2012 9:09 PM
To: arab-gen from net.bio.net
Subject: [Arabidopsis] mRNA and sRNA from seedlings
My lab has noticed a very poor efficiency for isolating both mRNA and small RNA from some tissues, while other tissues seem fine. We are using Invitrogen's (now Life Technologies) Trizol product. As far as we can tell, there are abundant mRNA and sRNA before the isolation, but the Trizol seems to select for long non-mRNA. Invitrogen says the tissue shouldn't matter, but experimentally we see that it does. Can anyone suggest an RNA prep that can work from ANY tissue (seedling, seed, etc...), isolate all small RNAs (20-25nt), and recover a high percentage of the longer mRNAs?
R. Keith Slotkin
The Ohio State University
500 Aronoff Laboratory
318 West 12th Ave.
Columbus, OH, 43210
slotkin.2 from osu.edu<mailto:slotkin.2 from osu.edu>
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