Most of us are not aware of RNA quantification methods and analysis and how it benefits the laboratories. This article gives an overview of how it works. RNA is prone to degradation as the presence of RNA is high in nature. If an RNA analysis is successful, it means performing at its best during the process of purification.
Choose the right analysis and quantification methods for the applications and samples involved to make the analysis a complete one. Some of the common ways for RNA quantification as well as analysis have been discussed here. Besides, the use of the right applications for every method and its benefits are highlighted.
RNA Quantification Is an Integral Step before RNA Analysis
Common ways to quantify RNA and methods to maximize each method are as follows.
UV Spectroscopy
A traditional method is applied to assess RNA purity and concentration and it’s known as UV spectroscopy. The percentage of absorbance of an RNA sample measures at 260 and 280 nm respectively. As far as the nucleic acid concentration is concerned, calculate it using the law of Beer-Lambert.
The law predicts a change in the process of absorbance with concentration. The equation assesses the purity of RNA. This method is widespread as most people apply it to quantify RNA. As far as the availability of UV spectrophotometers is concerned, there is no dearth as it’s available in every laboratory. There is no denying that this method also has some pitfalls, but you can reduce them with the help of the tips given below.
How to Maximize the Performance?
- The method does not differentiate between DNA and RNA. Therefore, it is best to go for RNA samples using RNAse-free for treatment and DNAse should remove contaminated DNA. Check out some of the sites that provide solutions to help users perform better analysis and readings with regards to RNA quantification. They also provide useful tips to ensure you reap maximum benefits out of it.
- Phenol, residual proteins, and other related contaminants come in the way of absorbance analysis. Hence, take extra measures while performing RNA purification to get rid of them.
- The reading samples use a quartz cuvette to perform analysis. Dust particles and cuvette lead to light scatter that impacts the absorbance. Perform a thorough background check to make readings using a blank.
- Ensure that the RNA dilution does not exceed the range of the spectrophotometer. You cannot measure the solutions that are out of range with accuracy. Most often, errors take place when there are lesser concentrations.
Importance of High-Quality RNA
To downstream the applications, quality RNA is a must. The requirement with regards to quality varies based on the applications. The RNA which is not suitable for a specific application offers the best outcomes to another. The higher-cost assays call for more attention and hence analyze RNA samples meticulously to avoid downstream failure. Also, reduce costs related to repetitive analysis. Interpret the data accurately from quality control and quantification analysis to ensure there are no pitfalls.
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