Determination of Amount of RNA by the Orcinol Method

• RNA
• Alkaline distilled water
• Acid-orcinol reagent
• Boiling water bath
• Spectrophotometer and cuvettes

1. Prepare a series of serially diluted RNA standards, but with a range from 1.0 mg/mL down to 0.125 mg/mL.
2. Prepare a serial dilution of your sample RNA.
3. Place 3.0 mL of each standard and 3.0 mL of each serial dilution of the sample RNA into separate test tubes. Place 3.0 mL of alkaline water in a separate tube.
4. Add 3.0 mL of acid-orcinol reagent to each tube and mix well.
5. Add 0.3 mL of alcohol-orcinol reagent to each tube and mix well.
6. Place the tubes in a boiling water bath for 20 minutes, with marbles placed on top of each tube to prevent evaporation. Cool the tubes by immersion in an ice bath at the end of the 20-minute period.
7. Turn on a spectrophotometer and adjust the wavelength to 660 nm. Blank the spectrophotometer with the alkaline water/orcinol tube. Measure the A₆₆₀ of each of the remaining standards and diluted samples.
8. Plot the absorbance of the standards against the known concentrations. Calculate the extinction coefficient, and calculate the concentration of RNA in your sample. Use the dilution yielding an absorbance between 0.1 and
   1.5 absorbance units.