Strange question on TRIzol and RNAlater - (Aug/30/2011 )
It is actually a solved problem, but I'm puzzled about the reason behind our solution.
Our lab does a lot of RNA extractions with RNAlater-treated tissues using TRIzol, but we have been plagued with a lack of consistency in RNA quality--which is especially an issue when we're going into RNA-seq.
I have then noticed that the RNAlater manual included the following:
at the precipitation step because of RNAlater carryover. If this occurs, simply add a mixture of
50% water, 50% isopropanol until the solution becomes clear and the two phases mix. The amount of
water/isopropanol required will depend on how much RNAlater was carried over; if the sample was
mostly RNAlater, as much as an equal volume may be needed.
RNAWIZ, of course, is no TRIzol, but is close enough that MRC stopped Ambion from selling it. So I asked Invitrogen CS if the above applies to TRIzol too, and they gave a affirmative answer. So I performed a test extraction this way:
2mL of TRIzol homogenate were split into 2X1mL aliquots. After phase separation, one aliquot will perform precipitation with 1 vol isopropanol, while the aqueous phase from the other aliquot was mixed with 0.5 vols of water, and then 1.5 vols(i.e. total volume of the diluted aquaous phase) of isoproranol. The extracted RNA were then ran on a non-denaturing gel.
The result was: samples that used the latter procedure has clearly stronger rRNA bands than ones that uses the standard protocol.
But why is that?
So both these samples had RNAlater in them?
If so, I suspect that the RNAlater is a strongly polar compound, so it mixes well with water but not isopropanol which is non-polar... This is just a guess though.
Otherwise it could be that it is heavily salted, in which case the IPA will not mix in so well, so your RNA won't precipitate out fully.
bob1 on Tue Aug 30 23:51:21 2011 said:
So both these samples had RNAlater in them?
If so, I suspect that the RNAlater is a strongly polar compound, so it mixes well with water but not isopropanol which is non-polar... This is just a guess though.
Otherwise it could be that it is heavily salted, in which case the IPA will not mix in so well, so your RNA won't precipitate out fully.
Well, one extra tidbit: these's no real difference between the RNA concentration, 260/280 or 260/230 between ones extracted using standard protocol and the ones extracted using the modified protocol.
From what I know, RNAlater is mainly ammonium sulphate.
So heavily salted then, which would result in it being harder for IPA to dissolve.
What you are seeing on the gel is part of the reason why you shouldn't just rely on a spectrophotometer to assess the integrity of the extraction. Degraded RNA or DNA will still give a concentration reading in a spec.
For some reason one of your extractions has resulted in more intact DNA. I can't say whether it is the result of the H2O/isopropanol thing or increased volume, or something else.