Does anyone have any suggestions to prevent RNAse contamination when working with RNA?
I tend to have issues with degradation regardless of whether I use DEPC treated / RNAse free water and filtered pipette tips.
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$\begingroup$ Are you working with isolated RNA or with in vitro transcribed RNA? $\endgroup$– Mad Scientist - on strikeDec 14, 2011 at 21:40
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$\begingroup$ @MadScientist: Isolated RNA with a Qiagen RNeasy Kit. $\endgroup$– GWWDec 14, 2011 at 21:43
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$\begingroup$ My answer is about avoiding RNAses while working with RNA, I'm not working with isolated RNA so there are probably additional steps you should take to avoid RNAses present during the isolation. $\endgroup$– Mad Scientist - on strikeDec 14, 2011 at 21:44
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$\begingroup$ @MadScientist: I still think your answer is very relevant, I could be getting degradation during the down-stream experiments as well. I was hoping that this question could be relatively general because I know a lot of different people have different processes to avoid RNAse contamination. $\endgroup$– GWWDec 14, 2011 at 21:48
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3 Answers
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You need to be careful about everything that comes in contact with your samples. Every spatula, beaker and every magnetic stir bar need to be RNAse-free.
For metal- and glassware we usually put everything into a drying closet at 200-250 °C for a few hours. That should get rid of RNAses pretty efficiently.
The RNA chemicals should be seperated from other chemicals if not everyone in the lab is working with RNA. They shouldn't be weighed using spatulas, but just poured into an RNAse-free container to avoid contaminating the storage container.
Use disposable plastic wherever possible, you should be able to perform most stuff in falcons and Eppendorf tubes.
Be careful with performing e.g. a DNA-prep at the same bench as the RNA work. The first buffer for the DNA-prep contains RNAse.
Avoid keeping your Eppendorf tubes or falcons open for long, that should reduce the potential for contamination.
We've had pretty good experience with just using water from a Millipore system and autoclave it once, that seems to get rid of the RNAses. I know that this is not a sure thing as the RNAses can survive autoclaving, but it seems to work well enough.
answered Dec 14, 2011 at 21:32
Mad Scientist - on strikeMad Scientist - on strike
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Here are some tips from what I routinely do:
- wipe all the surfaces (including pipettes and gloves) with RNAse Away or similar
- do everything on ice
- use a RNAse/DNAse free tubes. Use also commercial RNAse free water (it's worth the investment).
- keep all your RNA reagents separate from your cell culture/animal work reagents. Autoclave all your buffers and don't let them sit forever. Just make fresh buffers every now and then.
- don't talk over the tubes
If you keep having degradation following all the above, consider how you prepare your sample. Do you have enough RNAse inhibitors in there? Do you use a RNA-conserving solution? Is your tissue sitting for a long time before the lysis? If your extracting RNA from cells, try using trizol or a similar reagent to preserve the RNA right from the lysis. If your getting the RNA from tissue samples, try snap freezing them in liquid nitrogen.
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Apart from tips already mentioned, change your gloves every now and then.
Also, in my experience (with the Qiagen kit), keeping samples on ice shouldn't make much of a difference.
