It is common knowledge in most laboratories that RNA is more susceptible
to degradation than DNA. Unlike Deoxyribonucleases (DNases), which require
metal ions for activity (and can therefore easily be inactivated with chelating
agents such as EDTA), ribonucleases (RNases) have virtually no cofactor
requirements and advantage of the 2 hydroxyl groups adjacent to the
phosphodiester linkages in RNA as a reactive species.
Sources for RNase contamination in the Laboratory
It is possible to contaminate samples with RNases during the course of
an experiment in various ways:
- Autoclaving buffers and solutions will not inactivate RNases and may
actually introduce them into buffers e.g. by liberating them from contaminating
- Ungloved hands represent a source for introducing bacteria into solutions
resulting in RNase contamination and can even contaminate solutions
directly with released cellular RNases.
- Airborne contaminants settling on the surface of a solution of buffer
could also carry RNases.
Microbial ribonucleases share many properties with bovine pancreatic RNase
A. The pancreatic ribonucleases are small enzymes (15 kd) and consist of
a single polypeptide chain with four disulfide bridges. RNase A is a single-strand
specific endoribonuclease that is active over a wide pH range, is resistant
to metal chelating agents and can survive prolonged boiling or autoclaving
(1). RNase A-type enzymes rely on histidine residues within the active site
for catalytic activity and can be inactivated by the alkylating agent diethyl
pyrocarbonate (DEPC) which modifies these residues. RN
can effectively be avoided by using RNase-free solutions such as Eppendorf
Molecular biology reagents and following a few common sense laboratory procedures:
- Always wear gloves when working with RNA.
- Maintain a separate area for RNA work that has an own set of pipettes,
pipette tips, Eppendorf tubes, buffers, and reagents. This is especially
important if your work requires the use of RNases for e.g. plasmid preparations.
- Sterile disposable plasticware produced under clean room conditions
is RNase-free and should be used when possible. Eppendorf Biopur tips
and tubes provide this quality feature in addition to being DNase free.
- Metal tools like spatulas can quickly be decontaminated by holding
in a burner flame for several seconds. Contaminating RNases on glassware
can be inactivated by baking the glassware at 180C or higher for
Alternatively, glassware can be soaked in freshly prepared 0.1% DEPC in
water or ethanol for 1 hour, drained, and autoclaved (necessary to destroy
any unreacted DEPC which can otherwise react with other proteins and the
adenine residues of RNA). As DEPC will attack polycarbonate (e.g. centrifuge
tubes) or polystyrene (e.g. standard microtiter plates) decontamination
of these materials can be achieved by soaking in 3% hydrogen peroxide for
10 minutes. Residuals of pero xide can be removed by extensively rinsing
with RNase-free water1., 2.. An alternative protocol uses a 1 N NaOH soak
of 1 hour at 37C. After soaking in NaOH the labware is then washed
extensively in RNase-free water.
RNase decontamination of buffers and solutions
DEPC treatment of solutions is accomplishe
d by adding 1 ml DEPC per liter
of solution, stirring for 1 hour, and autoclaving for one hour to hydrolyze
any remaining DEPC into CO2 and Ethanol. Compounds with primary amine
groups (e.g. Tris) will react with DEPC. Consequently, Tris buffers should
be prepared by dissolving Tris base (from fresh bottle reserved for RNA
work) in DEPC-treated and autoclaved water, adjusting the pH (with an
electrode reserved for RNA work), and re-autoclaving to sterilize. Solutions
of thermolabile materials (e.g. DTT, nucleotides, manganese salts) should
be prepared by dissolving the solid (highest available purity) in DEPC-treated
and autoclaved water and passing the solution through a 0.2 m filter
to sterilize2., 3.. As an alternative to DEPC, which inhibits enzymatic
reactions and modifies both RNA and Tris if not completely hydrolyzed,
we recommend the use of RNase-free Molecular Biology Grade Water.
Use of ribonucleases inhibitors
A very convenient and effective way to protect RNA from decomposing RNase
activity is the use of an RNase inhibitor. Eppendorf Prime RNase Inhibitor
is a protein of non-human origin that binds non-covalently to RNase and
inhibits the same type of ribonucleases as human placental RNase inhibitors
(HPRI), including RNases A, B, and C. Prime RNase Inhibitor is stable
under a broad range of pH, DTT concentrations and temperatures. It inhibits
greater than 90% of RNase activity in reactions where an equal amount
of HPRI inhibits only 50%.
1. Blackburn, P., and Moore S. (1982) The Enzymes. Academic Press, NY,
2. Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning:
A Laboratory Manual. Cold Spring Harbor Press,
Cold Spring Harbor, NY,
3. Blumberg, D.D. (1987) Methods Enzymol. 152, 2024.
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