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Ribonucleic acid (RNA) quantification is a means of determining the average concentration of RNA in a solution. This determination can be performed using a variety of procedures, which usually fall into one of two categories: spectrophotometry or fluorescent dye quantification. Spectrophotometry relies on the ability of RNA to absorb certain wavelengths of ultraviolet light. Some fluorescent dyes, such as ethidium bromide, can bind to nucleic acids like RNA, and will fluoresce when they bind, allowing the luminosity to be measured.
When RNA is exposed to ultraviolet light, it will selectively absorb this light at the wavelengths of 260 nanometers (nm) and 280nm. This method is performed in a spectrophotometer, which produces wavelengths of ultraviolet light, and measures the light that passes through the RNA. Greater concentrations of RNA will absorb more light.
A combination of these two wavelengths is often used in RNA quantification since this method allows researchers to learn whether a sample is contaminated by other macromolecules, such as proteins. These contaminants will often selectively absorb 280nm light, but not light at 260nm. As a result, calculating the ratio of absorbed light at both wavelengths can determine the degree of contamination.
RNA quantification using fluorescent dyes gives results that are less susceptible to some contaminants, and can be used with low levels of RNA that would make spectrophotometry impossible. Dyes like ethidium bromide will bind to RNA, and the resulting luminosity can be measured directly using fluorescence photometers. If a photometer is not available, solutions with known concentrations of RNA can be prepared, and the unknown sample's luminosity can be roughly compared to these. The relationship between luminosity and RNA concentration is linear, so researchers can quickly determine a concentration measurement from luminosity.
RNA quantification using either method can be highly susceptible to different contaminants. Proteins, phenol, and large particulates can all make the results of spectrophotometry inaccurate. These contaminants do not affect fluorescent dye RNA quantification, but this method can be rendered inaccurate by the presence of deoxyribonucleic acid (DNA) in a sample.
Dyes that bind nucleic acids will bind both DNA and RNA, and exhibit similar luminosities, so ensuring a clean sample of RNA is important. The usual way to accomplish this is by adding an enzyme that destroys DNA, such as DNAse, to a mixed sample before adding a dye. Depending on the concentration of RNA in a sample, and which contaminants are present, laboratories may use either of these methods to quantify RNA.
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