Very thin aqueous layter (Trizol RNA extractions)

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Very thin aqueous layter (Trizol RNA extractions)

Postby Gradster28 » Nov 07 2015 5:57 pm

I'm trying to extract RNA from very small pools of insects. I'm using a trizol extraction protocol with either beads and a vortexer or just a mortar and pestle method to break up the tissue. I tested the 260/280 ratios of the first couple batches and it was pretty low, but I was told by the person who taught me how to do extractions that it was probably because I suspended the RNA in water instead of a buffer and that I shouldn't worry about it. I also often have a very small or invisible pellet, but was told that's probably because of the very small amount of tissue I started with. Again, I haven't been worrying. But, the last couple batches yielded an extremely thin aqueous layer. It's always been thin, but this time it was thin enough that avoiding the pink layer was extremely difficult.

I guess my question is, is it possible that this is another result of just having very small tissue samples or could there be something else wrong? Is there anything I can do to increase the aqueous layer for the next batch?
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Re: Very thin aqueous layter (Trizol RNA extractions)

Postby mchlbrmn » Nov 08 2015 1:11 pm

The size of the aqueous layer should be related to the amount of trizol, plus water from the sample, not directly proportional to the sample. The Trizol should be about 50:50 water and phenol (I actually don't use Trizol, but this should b e the general Chomczinski method I assume). They are miscible, forming one phase since the solution also contains guanidinium isothiocyanate and low pH sodium acetate. When some chloroform, or another chemical, is added, the organic chemicals become even more hydrophobic, and are no longer miscible with water. Then the organics separate from the aqueous and form another phase. The top aqueous phase should be about 50- 60% of the volume, I believe. At low pH the DNA will partition with the organics, and the RNA with the water. A bit more or less bug added shouldn't really change this. If insufficient, chloroform, or other agent, was added the phases may not separate as completely, and you may have only a small water phase. If this the case, then more chloroform would fix this. I believe the chloroform should be 20% of the Trizol volume.
I always resuspended my RNA in water. By the Chomczynski method my O.D.s were low, perhaps a260/280 about 1.6, if I recall. When I used column purification, the ratio was close enough 1.8. Whatever residual contamination lowered the ratio did not seem to be a problem with RT PCR generally.
If you must work with low volumes of water in an organic extraction, you can remove only what is safe to take without getting any organic phase, and then do a second back extraction by adding more water phase, vortexing, centrifuging, and removing a second time. I haven't done this (in memory) in an RNA xtraction, so you may need to test the procedure. Perhaps it's more ideal to use guanidinium isothiocyanate denaturing solution, as the original aqueous layer contains, to keep RNase inactive. Or, perhaps it's OK to use water for the short time until you precipitate? Well, I suppose you would have already removed the rNAse in the extraction, but if you back extract with simple water, you may have lowered the salt concentration, and this may (?) affect the precipitation with isopropanol that follows? If so, it may help to add 1/10 the volume of the added water of 3M Sodium acetate before precipitating, as is normally done in a nucleic acid precipitation. I assume the Trizol already contains sufficient salt, but this would compensate for extra water you add. It's been a long long time, but my fuzzy recollection is that adding guanidinium isothiocyanate overloads what is already in the organic phase, and water plus sodium acetate is the way to go for a back extraction.
However, the aqueous removed should really be about 1/2 the Trizol volume, so back extraction may be unnecessary.
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Re: Very thin aqueous layter (Trizol RNA extractions)

Postby r.rosati » Nov 11 2015 12:50 pm

I confirm that after phase separation, you should see at least 400ul of aqueous layer per ml of trizol.
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