Product Manual
www.bioline.com/isolate
20
3. how to maintain an rnase-free environment
•
Gloves: Always wear sterile gloves before handling any thing that is going to
be used for RNA analysis. It is however important to remember that once the
gloves have touched equipment in the lab such as centrifuges, pipettes and
door handles, they are no longer RNase-free.
•
Disposable plasticware: Disposable plasticware greatly reduce the possibility
of contaminating your samples. In the event of a contamination, they also
minimize the spread of the contamination. The use of disposable tips, tubes,
etc. is therefore highly recommended.
•
Good quality reagents: Always ensure that all reagents and chemical
purchased commercially are guaranteed to be RNase free. Testing each batch
before use may be a prudent step.
•
DEPC-treated water: Use DEPC-treated water instead of regular PCR grade
water. DEPC inactivates RNase by histidine modification of the bases. If
DEPC-treated water is made in-house, always remember to autoclave before
use to degrade the DEPC.
•
RNase inhibitors: The use of RNase inhibitors is highly recommended with
samples containing endogenous RNase. Most RNase inhibitors are suitable
for use in any application where RNases are a potential problem.
•
Decontamination techniques: Heat proof glassware can be baked at 180ºC
for several hours to inactive RNases. Polycarbonate or polystyrene materials
can be decontaminated by soaking in 3% hydrogen peroxide for 15 minutes,
followed by thorough rinsing with RNase-free water.
•
Correct storage of RNA is also very important to avoid RNA degradation. In
the short term, RNA may be stored in RNase-free H
2
O or TE buffer at -80ºC for
1 year without degradation. For long term storage RNA samples may be stored
as ethanol precipitates at -20ºC. However, when dissolved in ethanol, RNA is
not dispersed evenly in the solution and cannot be used directly in quantitative
experiments. Instead, precipitates should be pelleted and redissolved in an
aqueous buffer before pipetting.
