Biochemical Techniques and Instrumentation

RNA Quantification by Spectrophotometric Determination

Analysis of UV absorption by the nucleotides provides a simple and accurate estimation of the concentration of nucleic acids in a sample. Purines and pyrmidines in nucleic acid show absorption maxima around 260nm (eg., dATP: 259nm; dCTP: 272nm; dTTP: 247nm) if the DNA sample is pure without significant contamination from proteins or organic solvents. The ratio of OD₂₆₀/OD₂₈₀ should be determined to assess the purity of the sample. This method is however limited by the quantity of DNA and the purity of the preparation. Accurate analysis of the DNA preparation may be impeded by the presence of impurities in the sample or if the amount of DNA is too little. In the estimation of total genomic DNA, for example, the presence of RNA, sheared DNA etc. could interfere with the accurate estimation of total high molecular weight genomic DNA. It is possible to detect and quantify at concentrations as low as 2.5 ng/µl. The nitrogenous bases in nucleotides have an absorption maximum at about 260 nm. Using a 1-cm light path, the extinction coefficient for nucleotides at this wavelength is 20. Based on this extinction coefficient, the absorbance at 260 nm in a 1-cm quartz cuvette of a 50µg/ml solution of double stranded DNA or a 40µg/ml solution of single stranded RNA is equal to 1. We can calculate the concentration of the DNA or RNA in the sample as follows:

DNA concentration (µg/ml) = (OD ₂₆₀) x (dilution factor) x (50 µg DNA/ml)/(1 OD₂₆₀ unit)

RNA concentration (µg/ml) = (OD ₂₆₀) x (dilution factor) x (40 µg RNA/ml)/(1 OD₂₆₀ unit)

In contrast to nucleic acids, proteins have a UV absorption maximum of 280 nm, due mostly to the tryptophan residues. The absorbance of a DNA sample at 280 nm gives an estimate of the protein contamination of the sample. The ratio of the absorbance at 260 nm/ absorbance at 280 nm is a measure of the purity of a DNA sample; it should be between 1.65 and 1.85.

Phenol has an absorbance maximum of 270 but the absorbance spectrum overlaps considerably with that of nucleic acids. If there is phenol contamination in the DNA sample, the absorbance at 260 nm will be high, giving a false measure of DNA concentration.