Nucleic acid quantification

For quantification analysis spectrophotometry is recommended. This technique gives you information about the nucleic acid concentration of your sample together with two quality ratios, 260/280 and 260/230. To get information about RNA integrity other techniques need to be applied.

The 260/280 ratio reflects the purity of DNA and RNA. At 260 nm you measure nucleic acids and at 280 you measure proteins. The recommended 260/280 ratio should be between 1.8 and 2.1. If the ratio is lower, it may indicate the presence of proteins or other contaminants that absorb at 280 nm.

The 260/230 ratio is used as a secondary measure of nucleic acid purity. At 260 nm you measure nucleic acids and at 230 you measure chemicals remaining in your sample from the isolation step. The 260/230 values should be in the range of 2.0-2.2. If the ratio is lower than expected it may indicate the presence of contaminants, which absorb at 230 nm (i.e. phenol).

Spectrophotometer measurement.

Nucleic acid concentration can be determined by measuring their absorbance using a spectrophotometer such as Nanodrop.

Absorbance is measured at 260nm (A260) where nucleic acids absorbs light most strongly. The amount of light absorbed is proportionate to the amount of nucleic acids in the sample.

The relationship between the concentration and the absorbance is described by the Beer-Lambert Law: A = c * ε * L, which can also be written as c = A / (ε * L), with

• c : the nucleic acid concentration in molar.

• A : the absorbance in AU.

• ε : the wavelength-dependent molar extinction coefficient (also called molar absorptivity) in molar/cm.

• L : the pathlength in cm (for NanoDrop this value is 10 mm).

This image depicts the output of a spectrophotometric RNA quantification of a high-quality RNA sample.