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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:
Quote
With Ambion’s RNAWIZ™, there may be a problem getting the aqueous phase to mix with isopropanol
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.
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?
