Abstract: The concentration of RNA should be determined by measuring the absorbance at 260 nm (A260) in a spectrophotometer.

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. As discussed below, 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, especially if the RNA is to be recovered after spectrophotometry. 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 = 2.2 ml
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 = 0.75
Concentration of RNA sample = 40 x A₂₆₀ x dilution factor
                                        = 40 x 0.75 x 50
                                        = 1500 μg/ml
Total yield      = concentration x volume of sample in milliliters
                    = 1500 μg/ml x 2.2 ml
                    = 3300 μg = 3.3 mg RNA

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. 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, dilution of the sample in water is recommended 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.