I will be working on setting up a dedicated RNA lab for RNA extractions and other sample preparations. Please let me know what guides are there to make sure I am handling RNA carefully and any other information about working in an RNA lab. I have all the pippetes and the whole lab bench with items that will be used for RNA sample prep only. I also need to know about freezing and storage of RNA at 4C, -20C, and -80C, how long can the RNA be stored in each of these temperatures?
Posted 04 June 2014 - 08:03 PM
I'm a graduate student in an RNA lab. Here are some tips:
-For RNA work, use filter tips only. It prevents possible contamination entering from the pipette.
-Keep the bench you use for minipreps away from your bench for RNA work to avoid RNaseA contamination. If you need to extract genomic DNA, do that protocol at your biowork bench. It's not really the E.coli or other organisms you need to worry about. It's the RNaseA added to the buffers for minipreps and other DNA extraction procedures.
-We store our RNA at -20C for long-term storage and it's fine. Some other RNA labs store it at -80C. If you have the space, I guess -80C is better, but we only have one -80C and we need it more for glycerol stocks and tissue storage. More important than the temperature is to avoid freeze/thawing of RNA. Aliquot RNA extractions into a few tubes so that you only need to thaw it once ever.
-I don't store RNA at 4C ever. The only reason you would keep RNA at 4C would be if you were actively using it for an experiment. Some in vitro experiments that require long incubations may need RNA to be kept at 4C.
-You don't mention your background, so sorry if some of this is very basic. To check the quality of your RNA extractions, run a little bit of your total RNA (e.g. I usually run 1 uL from a 50 uL elution) on an agarose gel. Most RNA in cells is ribosomal RNA. You should see two clear bands from the higher MW rRNAs in the cell. If the bands are sharp and clear, that's excellent. If the top band is equally bright as the second band, even better. If the bands are smeary, you have RNA degradation. Keep your RNA on ice when you're working with it and make sure you use water/buffers/tubes/tips that are exclusively used for RNA work. That will help.
-RNA extractions also contain some DNA. If you are doing RT-PCR or qPCR or otherwise any work that involves looking at transcript levels, you need to add a DNase step after your RNA extraction, before your cDNA synthesis. We use RQ1 DNase from Promega; it's cheap and works well. You can check if your DNase step worked by having a control without reverse transcriptase when you make cDNA. If you still have DNA left over, you'll see a product in your PCR. If you got rid of the DNA, then all you will see are primer dimers.
-Set up new glassware that will ONLY be used for water. Autoclave the water using regular protocols (e.g. 20-30 min). NEVER use these bottles for liquid media or anything else. If you maintain this policy strictly, you can get away with using water without DEPC. We don't bother with DEPC and we never have RNase problems. You really don't need DEPC if you can avoid RNase contaminations in the first place.
-I wipe my bench down with 10% bleach before working on RNA extractions. I also wipe my pipettes with 75% ethanol (not bleach because that hurts the plastic).
-Buy some RNAseAWAY. It's a soapy cleaner for plastics to help remove RNase contaminations. You don't need to use it constantly. I wash my tube racks with it once every 2 months or so.
-Make sure your disposable plastics say "RNAse-free."
-Does the RNA you work with have any important structures you want to maintain? When we want RNA to fold properly after heating, we let it cool gradually. When we want to disrupt structure, we put it on ice immediately so it doesn't have time to fold correctly.
-If possible, consider getting a nanodrop with multiple wells. Make some wells for RNA work only. If other people in your lab ever use them for crude protein, they are going to introduce RNase contaminations.
Edited by glez, 04 June 2014 - 08:07 PM.
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