Here are some tips for keeping an RNase-free zone in your lab:
- Wear gloves and use sterile technique when handling RNA or reagents that will be used with RNA. The most common sources of RNase contamination are hands, bacteria or mold that could be lurking on dust particles or on glassware.
- Use sterile, disposable plastic ware. These products are typically RNase-free and don’t require treatment to inactivate RNases.
- Treat non-disposable glassware and plastic ware before you use it. For glassware, bake it at 250oC overnight (i.e., sterilize glassware in the hot air oven at 250oC overnight). For plastic ware, rinse it with 0.1N NaOH/1mM EDTA and then with diethyl pyrocarbonate (DEPC)-treated water.
- Reserve a set of chemicals and solutions needed for RNA isolation and analysis as “RNA ONLY” and keep them separate from reagents for other applications. Wear gloves whenever you handle these reagents, and use sterile labware to measure them.
- BEWARE! Autoclaving alone is not sufficient to inactivate all RNases. Solutions that you prepare in the lab should be treated by adding DEPC to 0.05% and incubated overnight. Autoclaving the treated solutions for 30 minutes will remove any trace of the DEPC from the solution.
- Only buffer and water stocks treated to be RNase-free should be kept in the RNA area of the lab.
- Your RNase-free zone should have dedicated laboratory equipment (gel boxes, centrifuges, e.t.c.), glassware and plastic ware.
- The RNase-free zone should be a relatively low-traffic area away from open windows and doors.
- Using RNase inhibitors will help prevent RNA degradation by inhibiting RNases from some common sources.
PRO TIP 1
- You can treat water with DEPC to inactivate RNase
- Recipe for 0.05% DEPC (v/v) solution: 50µl DEPC per 100ml solution.
- NOTE: DEPC is a suspected carcinogen. Take appropriate precautions.
PRO TIP 2
- Tris-based buffers cannot be treated with DEPC.
- Purchase Tris that is RNase-free.
- Alternatively, use DEPC-treated or nuclease-free water to make your Tris-based solutions.
Choosing an RNA Isolation System
There are several factors to consider when choosing an RNA purification system.
Source of RNA
- Tissues known to contain many RNA
- total RNA
- Limited, critical sample
For a full listing of the RNA purification systems, ranging from manual, single-tube purification systems to high-throughput options that can be run using a liquid handler system, always do well to visit the PROMEGA website @: www.promega.com