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I've gotten conflicting advice on this: some people believe one can remove RNase contamination simply by spraying the bench, pipettes, gloves, etc. with ethanol. Others think ethanol does not destroy RNase and special reagents are needed to prevent RNase contamination.

So does ethanol remove RNase contamination? If not, what reagent does?

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See the answer from shigeta below - "inhibition" isn't really the right way to think about this, since that may imply a specific interaction when talking about an enzyme. –  Alan Boyd Feb 16 '13 at 8:03
    
Good point, I changed the wording of the question to convey the need for permanent denaturation. –  Drosophila Feb 16 '13 at 11:30
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2 Answers 2

up vote 5 down vote accepted

I think the protocols to clean glassware take no chances and just care about RNA degradation rather than targeting one class of enzymes.

EtOH is supposed to denature RNAse and any other proteins on the surface.

Other chemicals such as Diethylpyrocarbonate (DEPC) used to wash glassware kill all biochemical activity by reacting with nucleophilic moieties in the protein (-OH, -SH -NH(2/3) ). (See comments below for more.)

These treatments are followed by heating at 300-450 F for 2-4 hours. This is not a specific RNAse decontamination - all biological activity is killed.

RNAse activity is not assessed by specific detection of a particular enzyme, but rather judged by the degradation of an RNA sample in the lab environment. So specific inhibition isn't worthwhile - any protein that might interact with and degrade RNA is called an RNAse in these protocols.

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In short, RNA contamination and degradation are super easy in a lab setting. We have an RNA-only dedicated workbench in our lab which gets treated with a "broad spectrum" RNAse-degrading solution before any RNA work is conducted. –  leonardo Feb 16 '13 at 3:15
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The heat treatment is called depyrogenation and not only completely removes all biological activity, it also destroys pyrogens (fever inducers) such as LPS or endotoxin. –  MattDMo Feb 16 '13 at 3:25
    
Thanks for adding to this guys. I think you can see how the reagents really clean up the glass. I'm familiar with the protocols, but I've not worked with RNA myself. –  shigeta Feb 16 '13 at 3:42
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You don't want to only inhibit or temporarily denature RNAses, if you work with RNA you have to permanently inactivate the RNAses. I work with RNA, and I haven't seen anyone use ethanol to remove RNAses, I would not trust it to work reliably. RNAses are really stable enzymes, they even survive autoclaving to some part, so I would not be suprised if they can refold after ethanol exposure.

The usual methods to remove RNAses are

  • Treatment with 0.1% DEPC (heat up to at least 60 °C, or autoclave if possible to remove residual DEPC later). Cannot be used with e.g. Tris buffer or anything else that reacts with it. DEPC is carcinogenic, so you should follow the proper safety procedures.
  • Anything made of metal or glass, heat up to 250 °C for around 2 hours
  • 1M NaOH for at least 30 minutes (you need to wash thoroughly to remove the NaOH)

You should be aware that anything that destroys RNAses will likely do the same to your RNA.

Autoclaving does not protect completely against RNAses, but it still reduces RNAse activity significantly. So while you should not rely on autoclaving to get rid of RNAses, in my experience it is sufficient if you start from a source that is unlikely to contain a lot of RNAses. Pipette tips (not loose tips out of a bag, to minimize handling) and millipore-filtered water are pretty safe after they are autoclaved.

There are also commercial RNAse inhibitors, but I've only used them in cases where I had to mix RNA and protein, and the protein wasn't completely RNAse-free.

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