General Guidelines for Working
with RNA
Handling
RNA
Ribonucleases
(RNases) are very stable and active enzymes that generally do not require cofactors
to function. Since RNases are difficult to inactivate and even minute amounts
are sufficient to destroy RNA, do not use any plasticware or glassware without
first eliminating possible RNase contamination. Great care should be taken to
avoid inadvertently introducing RNases into the RNA sample during or after the
isolation procedure. In order to create and maintain an RNase-free environment,
the following precautions must be taken during pretreatment and use of
disposable and non-disposable vessels and solutions while working with RNA.
General
handling
Proper
microbiological, aseptic technique should always be used when working with RNA.
Hands and dust particles may carry bacteria and molds and are the most common
sources of RNase contamination. Always wear latex or vinyl gloves while
handling reagents and RNA samples to prevent RNase contamination from the
surface of the skin or from dusty laboratory equipment. Change gloves
frequently and keep tubes closed whenever possible. Keep isolated RNA on ice
when aliquots are pipetted for downstream applications.
Disposable
plasticware
The use of
sterile, disposable polypropylene tubes is recommended throughout the
procedure. These tubes are generally RNase-free and do not require pretreatment
to inactivate RNases.
Non-disposable
plasticware
Non-disposable
plasticware should be treated before use to ensure that it is RNase-free. Plasticware
should be thoroughly rinsed with 0.1 M NaOH, 1 mM EDTA followed by RNase-free
water. Alternatively, chloroform-resistant plasticware can be rinsed with
chloroform to inactivate RNases.
Glassware
Glassware
should be treated before use to ensure that it is RNase-free. Glassware used for
RNA work should be cleaned with a detergent, thoroughly rinsed, and oven baked
at 240°C for four or more hours (overnight, if more convenient) before use.
Autoclaving alone will not fully inactivate many RNases. Alternatively,
glassware can be treated with DEPC[1]
(diethyl pyrocarbonate). Fill glassware with 0.1% DEPC (0.1% in water), allow
to stand overnight (12 hours) at 37°C, and then autoclave or heat to 100°C for
15 minutes to eliminate residual DEPC.
Electrophoresis
tanks
Electrophoresis
tanks should be cleaned with detergent solution (e.g., 0.5% SDS), thoroughly
rinsed with RNase-free water, and then rinsed with ethanol* and allowed to dry.
Solutions
Solutions
(water and other solutions) should be treated with 0.1% DEPC. DEPC is a strong,
but not absolute, inhibitor of RNases. It is commonly used at a concentration
of 0.1% to inactivate RNases on glass or plasticware or to create RNase-free
solutions and water.
DEPC
inactivates RNases by covalent modification. Add 0.1 ml DEPC to 100 ml of the solution
to be treated and shake vigorously to bring the DEPC into solution. Let the solution
incubate for 12 hours at 37°C. Autoclave for 15 minutes to remove any trace of DEPC.
DEPC will react with primary amines and cannot be used directly to treat Tris buffers.
DEPC is highly unstable in the presence of Tris buffers and decomposes rapidly into
ethanol and CO2. When preparing Tris buffers, treat water with DEPC first, and
then dissolve Tris to make the appropriate buffer. Trace amounts of DEPC will
modify purine residues in RNA by carboxymethylation. Carboxymethylated RNA is
translated with very low efficiency in cell-free systems. However, its ability
to form DNA:RNA or RNA:RNA hybrids is not seriously affected unless a large
fraction of the purine residues have been modified. Residual DEPC must always
be eliminated from solutions or vessels by autoclaving or heating to 100°C for
15 minutes.
Storage
of RNA
Purified RNA
may be stored at –20°C or –70°C in water. Under these conditions, no degradation
of RNA is detectable after 1 year.
Quantitation
of RNA
The
concentration of RNA should be determined by measuring the absorbance at 260 nm
(A260) in a spectrophotometer. To ensure
significance, readings should be greater than 0.15. An absorbance of 1 unit at
260 nm corresponds to 40 µg of RNA per ml (A260 =1= 40 µg/ml).
This relation is valid only for measurements in water. Therefore, if it is
necessary to dilute the RNA sample, this should be done in water. The ratio
between the absorbance values at 260 and 280 nm gives an estimate of RNA
purity. When measuring RNA samples, be certain that cuvettes are RNase-free.
This can be accomplished by washing cuvettes with 0.1M NaOH, 1 mM EDTA followed
by washing with RNase-free water. Use the buffer in which the RNA is diluted to
zero the spectrophotometer.
An example of
the calculation involved in RNA quantitation is shown below:
Volume of RNA
sample = 100 µl
Dilution = 10
µl of RNA sample + 490 µl distilled water (1/50 dilution).
Measure
absorbance of diluted sample in a 1 ml cuvette (RNase-free). A260 = 0.23
Concentration
of RNA sample = 40 x A260 x dilution factor= 40 x 0.23
x 50 = 460 µg/ml
Total yield =
concentration x volume of sample in milliliters= 460 µg/ml x 0.1 ml= 46 µg
Purity
of RNA
The ratio of
the readings at 260 nm and 280 nm (A260/A280) provides
an estimate of the purity of RNA with respect to contaminants that absorb in
the UV, such as protein. However, the A260/A280 ratio is
influenced considerably by pH. Since water is not buffered, the pH and the
resulting A260/A280 ratio can vary greatly. Lower
pH results in a lower A260/A280 ratio and
reduced sensitivity to protein contamination[2].
For accurate values, we recommend measuring
absorbance in 10 mM Tris·Cl, pH 7.5. Pure RNA has an A260/A280 ratio of 1.9–2.1
in 10 mM Tris·Cl, pH 7.5. Always be sure to calibrate the spectrophotometer with
the same solution.
For
determination of RNA concentration, however, we still recommend dilution of the
sample in water since the relationship between absorbance and concentration (A260 reading of
1 = 40 µg/ml RNA) is based on an extinction coefficient calculated for RNA in
water
DNA
contamination
No currently
available purification method can guarantee that RNA is completely free of DNA,
even when it is not visible on an agarose gel. To prevent any interference by
DNA in RT-PCR applications, we recommend designing primers that anneal at
intron splice junctions so that genomic DNA will not be amplified.
Alternatively, DNA contamination can be detected on agarose gels following
RT-PCR by performing control experiments in which no reverse transcriptase is
added prior to the PCR step or by using intron-spanning primers. For sensitive
applications, such as differential display, or if it is not practical to use
splice-junction primers, DNase digestion of the purified RNA with RNase-free
DNase is recommended.
Integrity
of RNA
The integrity and size distribution of total RNA purified with RNeasy Kits can be checked by denaturing agarose gel electrophoresis and ethidium bromide staining. The respective ribosomal bands should appear as sharp bands on the stained gel.
[1] DEPC is a suspected carcinogen and should be handled with
great care. Wear gloves and use a fume hood when using this chemical
[2] Wilfinger, W.W., Mackey, M., and Chomczynski, P. (1997) Effect of pH and ionic strength on the spectrophotometric assessment of nucleic acid purity. BioTechniques 22, 474.