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RNeasy Mini Handbook 10/2019
77
When measuring RNA samples, be certain that cuvettes are RNAse-free, especially if the RNA is
to be recovered after spectrophotometry. This can be accomplished by washing cuvettes with 0.1
M NaOH, 1 mm EDTA,* followed by washing with RNAse-free water (see “Solutions”, page 74).
Use the buffer in which the RNA is diluted to zero the spectrophotometer. An example of the
calculation involved in RNA quantification is shown below:
Volume of RNA sample = 100 µl
Dilution = 10 µl of RNA sample + 490 µl of 10 mm Tris·Cl,* pH 7.0 (1/50 dilution)
Measure absorbance of diluted sample in a 1 ml cuvette (RNAse-free)
A
260
= 0.2
Concentration of RNA sample = 44 µg/ml x
A
260
x dilution factor
= 44 µg/ml x 0.2 x 50
= 440 µg/ml
Total amount = concentration x volume in milliliters
= 440 µg/ml x 0.1 ml
= 44 µg of RNA
Purity of RNA
The ratio of the readings at 260 nm and 280 nm (
A
260
/
A
280
) provides an estimate of the purity
of RNA with respect to contaminants that absorb in the UV spectrum, such as protein. However,
the
A
260
/
A
280
ratio is influenced considerably by pH. Since water is not buffered, the pH and
the resulting
A
260
/
A
280
ratio can vary greatly. Lower pH results in a lower
A
260
/
A
280
ratio and
reduced sensitivity to protein contamination.† For accurate values, we recommend measuring
absorbance in 10 mm Tris·Cl, pH 7.5. Pure RNA has an
A
260
/
A
280
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 used
for dilution.
*
When working with chemicals, always wear a suitable lab coat, disposable gloves, and protective goggles. For more
information, consult the appropriate safety data sheets (SDSs), available from the product supplier.
† 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.
‡
Values up to 2.3 are routinely obtained for pure RNA (in 10 mM Tris·Cl, pH 7.5) with some spectrophotometers.
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