Ultraviolet absorbance at 260 and 280 nm in RNA measurement is dependent on measurement solution

RNA measurement is conducted by measuring ultraviolet absorbance at 260 nm and 280 nm. Calculation of the RNA concentration is based on the absorbance at 260 nm. Furthermore, RNA purity is judged as the 260 nm/280 nm ratio and a low ratio indicates contamination by protein. Diethyl-pyrocarbonate (DEPC)-treated water is used to dissolve RNA and 2-amino-2-hydroxymethyl-1,3-propandiol (Tris) is frequently added to the RNA dissolving solution in order to stabilize the RNA. In the present study, RNA was isolated from mouse liver, and then the influence of DEPC-treated water and Tris-buffer on RNA measurement was studied. The 260 nm/280 ratio of RNA determined after diluting it with distilled water was 1.82+/-0.01 (n=5). DEPC-treated water did not affect the absorbance at 260 nm, but elevated that at 280 nm. Thus, the 260 nm/280 nm ratio was as low as 1.52+/-0.01 (n=5). Tris-HCl (1 M, pH 7.0 or 10.0) lowered the absorbance at 260 nm and even more at 280 nm. Thus, the 260 nm/280 nm ratio was elevated to more than 2.17 (n=5). The present results clearly showed the influence of the measurement solution on RNA measurement.