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-   -   Total RNA stability following extraction/isolation (http://seqanswers.com/forums/showthread.php?t=24008)

pig_raffles 10-11-2012 01:03 AM

Total RNA stability following extraction/isolation
 
Hi all,

I will be extracting total RNA from animal tissue using the RNeasy minikit. The final step of which is to elute in to RNase-free water.

The next step, following extraction/isolation, will be to gauge the quality/quantity before sending away for library preparation and RNA-seq.

I have seen that some researchers use either RNA stabilizing solutions (e.g. RNA secure) instead of water or add RNase inhibiting supplements to the RNase-free water.

What do people use on this forum? What are the pros and cons of replacing/adding to RNAse-free water? Are there any downstream considerations to take into account, for example do some of these chemicals inhibit some of the reactions taking place in library preparation or sequencing?

Thanks

pmiguel 10-11-2012 10:10 AM

We add RNAse inhibitor to all RNA samples submitted to our core. The only concern I would have would with RNAse digestions in the first step of a procedure. Otherwise I trust the reaction clean-up methods to remove the inhibitor.

--
Phillip

pig_raffles 10-15-2012 06:35 AM

Quote:

Originally Posted by pmiguel (Post 86343)
We add RNAse inhibitor to all RNA samples submitted to our core. The only concern I would have would with RNAse digestions in the first step of a procedure. Otherwise I trust the reaction clean-up methods to remove the inhibitor.

--
Phillip

Hi Phillip,

Thanks for replying, could you tell me which RNase inhibitor you use and do you elute your samples in RNase-free water?

pmiguel 10-15-2012 06:55 AM

I would have to check to be sure, but I think we just use Promega RNAsin.

We use RNase-free water for pretty much everything. We actually have a reverse osmosis 18+ megaohm/cm water purifier that does UV treatment of the water it generates.

Alternatively Illumina assures us that "PW1" is nuclease free water. If you run an instrument, you likely have quite a bit of that on hand.

--
Phillip

ETHANol 10-16-2012 04:26 AM

I've left human total RNA purified with RNease on my bench for a few days and run it on the Bioanalyzer and there was no detectable degradation (no it wasn't on purpose). But it might depend on where you RNA is coming from.

yaximik 10-16-2012 04:52 AM

RNA isolated with RNeasy kit is pretty stable at -20, that is if no RNAse is brought along the way, which is obviously the key issue. A slightly acidic pH, like 5-6 with a few mM sodium citrate helps too. If RNA is concentrated, the best way for me was just storing it in pure formamide diluting to working concentration in water. A few % fomamide in solution does not seem to hurt enzymatic activities. Also, RNA can be eluted directly from glass fiber with fomamide instead of water.

pig_raffles 10-16-2012 04:52 AM

Quote:

Originally Posted by ETHANol (Post 86721)
I've left human total RNA purified with RNease on my bench for a few days and run it on the Bioanalyzer and there was no detectable degradation (no it wasn't on purpose). But it might depend on where you RNA is coming from.

So this was purely total RNA dissolved in RNase-free water at room temperature?

pmiguel 10-16-2012 05:28 AM

I think you want to fixate on Ethan's 2nd sentence:

Quote:

But it might depend on where you RNA is coming from.
Sure, pure RNA under mildly acid pH is stable at room temp. But those 2' hydroxyls are just aching to react with their adjacent phosphodiester bonds causing the formation of a cyclic '2-3' phosphate bond and cleaving the RNA polymer at that site. Increase the pH, the temp, add divalent cation, or, of course, an RNAse and it is goodbye intact RNA.

RNAse is a big problem both because there are likely multiple species of it present in every cell and your lab is likely replete with RNAseA (from the last plasmid or genomic DNA prep you or your colleague did) in the dust raining down on your bench.

--
Phillip


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