RNA quantification is done to determine the average level of concentration of ribonucleic acid in a given mixture. It is also useful in determining the purity of the nucleic acid. RNA quantification is a requirement to be fulfilled before the analysis of RNA is done. There are many methods to quantify RNA. Some of the common methods are discussed below:
#1 UV spectroscopy:
The most common method of RNA Quantification is UV spectroscopy. Under this method, the concentration of RNA is determined using a law called “Beer-Lambert.” This method is simple to perform, and most of the laboratories are equipped with UV spectrometers. While using this method to perform RNA Quantification, care should be taken to remove the residual proteins and phenol properly since they can interfere with the absorbance reading. To have an accurate reading of the RNA quantification, the dilution of RNA must fall within the linear range of the spectrophotometer. To have accurate measurements, the absorbance value has to fall between 0.1 and 1.0.
#2 Agilent 2001 Bioanalyzer:
This method uses a combination of various factors like a fluorescent dye, microfluidics, and capillary electrophoresis. These factors bind to the acid. This is how the concentration of RNA and its purity or RNA quantification is done under the method, “Agilent 2001 Bioanalyzer”. In this method, the determination of integrity is very accurate. In order to avoid underestimation of RNA concentration, the nucleic acid should be released into nuclease-free water. This can reduce the differences to some extent. The fluorescent dye used as one of the elements in the combination is sensitive to light. Hence, we must keep it away from light sources.
#3 Ethidium Bromide Fluorescence Emission:
Ethidium bromide is the most common type of dye used in RNA Quantification. Like mentioned in the previous method, this dye also binds to the RNA. Ethidium bromide can bind to both single and double-stranded molecules. This is a fast and simple procedure, and it can be performed with minimum volumes of sample. An instrument TurnerBiosystems TD 700 is used in the process. As already specified, it uses a combination of various elements. These elements are measured at room temperature in the instrument called biosystems TD 700.
When it comes to quantification of RNA, this method remains of high standard. This provides an unmatched level of accurate detection. To detect RNA, relevant primer sets must be designed. To make sure that the amplification is from the RNA and not from any other host, controls must be employed.
The integrity and purity of RNA:
The determination of the purity of RNA is quite simple. The OD values obtained during the routine spectrophotometry can be reviewed to evaluate the level of purity. But, this can be affected by a lot of external factors like the buffer pH, reference blank solution, proteins, ethanol, buffer salts, etc.
Thus, the above-discussed methods are some of the common methods used to perform RNA quantification to determine the level of concentration and the level of purity of the ribonucleic acid in any mixture under question.