Measuring RNA quality and quantity with the Nanodrop

There is an article from  Biomedial Genome’s webpage about RNA control quality which is a totally recommended reading. I’ve just copied the key information useful for us here.

There are three ways to determine RNA quality:

  • Measure the quantity;
  • Determine the purity;
  • Check the integrity;

Measuring the Quantity of RNA using the Nanodrop.

Nucleic acids are traditionally quantified using UV absorption using a spectrophotometer at 260 and 280 nm. RNA has its absorption maximum at 260 nm. We can use the ratio of the absorbance at 260 and 280 to assess RNA purity. Pure RNA has an OD A260/A280 ratio of 2.1. In many protocols, a value of 1.8-2.0 is acceptable.

When analysing RNA with the Nanodrop it is important to use exactly the same buffer as a diluent and as the blank, sometimes pure water is used incorrectly as a blank. Most protocols only mention the OD A260/A280 ratio but when you see a strong absorbing contaminant in the region of 230 nm this RNA is not as pure as RNA that has a single peak with a maximum at 260 nm. There are three sources of contaminations that produce peaks in the 220-230 nm region:

  • proteins
  • chaotropic salts like guanidinium isothiocyanate
  • phenol

All these three are either present in your tissue sample or are present in most tissue lysis solutions derived from the original Chomczynski and Sacchi protocol 1.

It is important that not only the OD A260/A280 ratio should be very close to 2.0, but that in addition, also the OD A260/A230  ratio should be very close to 2.0. Specially, when isolating low amounts of RNA the OD A260/A230 ratio drops significantly to sometimes under the 1.0. This clearly indicates, contamination with chaotropic salts or rests of phenol or protein in the RNA solution.

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  1. Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–9. 

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