Working with RNA? What fun! Those little, nearly indestructibleRNases are everywhere – on your skin and mucous membranes, in the waterand (some of the) enzymes you use, on lab surfaces, even in airbornemicrobes! Here are 10 ways to keep the RNases at bay, and keep yourprecious samples safe:
1. Clean everything; bench surfaces, pipettes,electrophoresis equipment and anything else you can think of with anRNase cleaning product, such as RNaseZap from Ambion(or 0.5% SDS followed by 3%H2O2). Establish a regular cleaning routine;a quick daily clean and a deeper weekly or monthly clean… and stick toit.
2. Treat your solutions. Good old DEPC is a fineway to keep your solutions RNase free. USe 0.5 mL DEPC/L, incubate for2 hr, autoclave for 45 minutes minimum. DMPC can also be used and maybe be safer than DEPC, which is a known carcinogen. Alternatively, manyvendors offer certified nuclease-free water, which may be worth theinvestment. Note that ultrafiltered water is already RNase free so does not need DEPC treatment. Also, don’t use DEPC/DMPC on tris-based solutions.
3. Designate a workspace, and a set of pipettes, if possible, that are dedicated to RNase-free work.
4. Use barrier tips. Barrier tips stopcross-contamination of your reagents and samples by preventing aerosolsreaching the barrel of your pipette. They are a must-have for RNA work.
5. Wear gloves and a lab coat. The obvious ones arethe best. Gloves and a lab coat will stop you from contaminating yoursamples with your own RNases. Change both frequently (maybe once perweek for lab coats). Also, when you have your gloves on don’t touchanything that is not decontaminated – door handles, taps, yourself… orother people (!).
6. Bake your glasswear. No enzyme can withstand baking for 300°C for 2 hours, but your glasswear can.
7. Isolate RNA using a method that eliminates endogenous RNAses, such as AquaRNA from Multitarget Pharmaceuticals, which is both clean and convenient.
8. Use RNase-free enzymes. Enzymes isolated frombacteria (e.g. DNase) can be full of RNase. Make sure you use certifiedRNase-free enzymes on your RNA samples where possible.
9. Use an RNase inhibitor when it’s not possible to keep things completely RNase-free. Roche’s Protector is a good example. Avoid high temperatures (above 60°C) or denaturing conditions that could deactivate the inhibitor!
10. Store RNA in ethanol at -80°C. Make aliquotsif the sample is to be used a number of times to avoid freeze/thawcycles. Before use, centrifuge to pellet the RNA, air dry thenresuspend in an RNase-free buffer.
11. Be completely paranoid, work as far away from your colleagues as possible, and shower in RNaseZAP five times per day. Just kidding.
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