RNA can be quantified through spectrophotometry or fluorescent dyes. Spectrophotometry measures the absorption of ultraviolet light, while fluorescent dyes bind to RNA and produce measurable brightness. Both methods can be affected by contaminants, but adding a DNA-destroying enzyme can ensure a clean RNA sample for accurate results.
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, usually falling into one of two categories: spectrophotometry or quantification of fluorescent dyes. Spectrophotometry is based on the ability of RNA to absorb certain wavelengths of ultraviolet light. Some fluorescent dyes, such as ethidium bromide, can bind to nucleic acids such as RNA and fluoresce when they bind, allowing the brightness to be measured.
When RNA is exposed to ultraviolet light, it will selectively absorb this light at wavelengths of 260 nanometers (nm) and 280 nm. This method is performed in a spectrophotometer, which produces wavelengths of ultraviolet light and measures the light passing through the RNA. Higher concentrations of RNA will absorb more light.
A combination of these two wavelengths is often used in RNA quantification as this method allows researchers to know if a sample is contaminated with other macromolecules, such as proteins. These contaminants often selectively absorb 280 nm light, but not 260 nm light. Consequently, calculating the ratio of absorbed light at both wavelengths can determine the degree of contamination.
Quantification of RNA using fluorescent dyes provides results that are less susceptible to some contaminants and can be used with low levels of RNA that would make spectrophotometry impossible. Dyes such as ethidium bromide bind to RNA and the resulting brightness can be measured directly using fluorescence photometers. If a photometer is not available, solutions with known concentrations of RNA can be prepared and the brightness of the unknown sample roughly compared to these. The relationship between brightness and RNA concentration is linear, so researchers can quickly determine a measurement of concentration from brightness.
RNA quantification using either method can be highly susceptible to different contaminants. Proteins, phenol, and large particles can cause spectrophotometry results to be inaccurate. These contaminants do not affect the RNA quantification of the fluorescent dye, but this method can be made inaccurate by the presence of deoxyribonucleic acid (DNA) in a sample.
Dyes that bind nucleic acids will bind both DNA and RNA and show similar brightnesses, so it is important to ensure a clean RNA sample. The normal way to accomplish this is to add a DNA-destroying enzyme, such as DNAse, to a mixed sample before adding a dye. Depending on the concentration of RNA in a sample and the contaminants present, laboratories can use one of these methods to quantify RNA.
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