Direct RNA Quantification
November 20, 2012Download
Related Products: Gen5 for Detection
Gen5 version required: 1.0 or higher
Basis for the Assay:
The aromatic ring structure of the purine and pyrimidine moieties that make up the nucleoside bases of DNA and RNA are responsible for absorbance of UV light at 260 nm. Although each specific base has a maximal absorbance at a slightly different wavelength, on average, nucleic acids as a macromolecule will absorb maximally very near 260 nm. In terms of quantitation, different nucleic acid species have different average extinction coefficients. By converting these known extinction coefficients to a specific concentration for a particular absorbance value at a defined wavelength, the nucleic acid concentration of solutions can be determined by a simple absorbance measurement. For example, the extinction coefficient for RNA (1 mg/ml) at 260 nm is 16 ODs for a 1 cm pathlength; this can be recalculated to mean 1.0 OD has a concentration of 40 μg/ml.
The protocol calls for an absorbance measurement at 260 nm along with a pre-read of the microplate to subtract background absorbance of the microplate. Pathlength correction is used to correct the absorbance value to reflect a pathlength of 1 cm. The corrected absorbance is then used to calculate RNA concentration.
The experiment file contains all of the absorbance determinations necessary including the pre-read plate (260 blank), the absorbance determinations at 977 nm (Test) and 900 nm (Reference), which are utilized to correct for pathlength, as well as the 260 nm experimental determination.
Plate Configuration: Plate layout includes samples in duplicate. There are no blanks or controls.
Report: The report contains a matrix with: 1. Raw absorbance values 2. Absorbance corrected to 1 cm pathlength 3. Calculated RNA concentration. The report also contains a table with the corrected ODs, calculated concentrations and standard deviation and % CV of the replicate sets.