How to Calculate RNA Concentration

••• Calculator image by Alhazm Salemi from Fotolia.com

Quantitate your RNA sample by measuring its absorbance of ultraviolet light (UV). A nano-drop spectrophotometer will use only one or two microliters of your sample, which you can recover. Other spectrophotometers require a much larger sample. The extinction coefficient for nucleotides at a UV wavelength of 260nm in a 1-cm light path is 20. Based upon this extinction coefficient, the absorbance of 40µg/ml RNA under the same conditions is one. Using this information, you can compute the concentration of your RNA sample.

    Make a dilution, if necessary, of your sample. A standard dilution for a microcuvette is 1:40. Make this dilution by adding 2µL RNA sample to 78µL sterile water.

    Follow the protocols of your particular spectrophotometer to calibrate the machine by using a blank and then determine the optical density of your sample at a UV wavelength of 260nm.

    Multiply the absorbance of your sample by your dilution factor by 40μg RNA/mL . The equation would be: “RNA concentration (µg/ml) = (OD260) x (dilution factor) x (40µg RNA/ml)/(1 OD260 unit)” (Hofstra.edu) For example: If you diluted your sample by 1:40 and your absorbance reading was 0.08, you would multiply 0.08 x 40 x 40 = 128 µg/ml = 0.13 µg/μL

    Figure out the purity of your sample by taking another absorbance reading at 280nm UV wavelength. The ratio OD 260/ OD 280 will indicate whether--and at what level--your sample is contaminated with protein or phenol. A result of 1.8 to 2.0 indicates quality RNA.

    Tips

    • Don't forget to calibrate your Spectrophotometer. Running a quick electrophoretic gel will confirm your spec results.

    Warnings

    • Don't assume your sample is pure. Taking the time for an OD260/OD280 ratio saves time and money down the road.

References

About the Author

Anne Mullenniex has been writing for eHow since 2009. She has owned and operated computer, construction, and insurance businesses. She has a bachelor's and a master's degree in biology and has taught at a community college. She is now working as an artist/artisan in multiple media.

Photo Credits

Dont Go!

We Have More Great Sciencing Articles!