The process of RNA isolation, purification and separation from samples is known as RNA extraction (1). Extraction of RNA is the requirement of many types of research and process. In many molecular techniques, extracting RNA is the most critical and important step. To make many processes successful, high-quality RNA extraction is a must.
This blog discusses the basics of extraction, collection, purification, and separation of RNA. The blog will also discuss some of the factors that will help to increase the quality of extracted RNA. RNA extraction procedures usually take place when RNase inhibitory agents are present.
SAMPLE COLLECTION AND PROTECTION:
To maximize the quality and yield of RNA preparation, finding the best method of cell disruption is crucial. During disruption of RNA isolation, denaturants must come in contact with cells at the time when the cells are disrupting. This may be a hassle in many ways. It could cause problems if cells are complex, if walls and capsules are contained, or if samples are large in number. This problem can be solved if we freeze cells in liquid nitrogen.
There are many methods available for nucleic acid purification and preparation. These methods can be classified into four general techniques.
- Organic extraction
- Spin basket format
- Magnetic particle
- Direct lysis
We can use all of these methods to prepare a good quality RNA, but certain things to keep in mind when you select the preparation technique. These factors are discussed below:
- Can you efficiently manage the sample?
- How much sample is required to process?
ISOLATED RNA QUANTITATION:
This step is crucial and necessary. At this step, we find out the average concentration of RNA in the given sample. To calculate the precise amount of RNA in a sample, the purity level of the sample is an essential factor to consider. There are three methods to discuss in this section:
- UV spectroscopy
- Fluorescent Dyes
- Agilent 2100 Bioanalyzer Instrument
This is the oldest and traditional method for RNA quantification. The absorbance of the sample is calculated at 260 and 2800 nm. The acid amount is measured using Beer-Lambert law. This law predicts the linear change in the absorbance with the amount of RNA.
We can use a fluorometer, filter fluorometer or fluorescence reader to detect the concentration of RNA with the help of RiboGreen reagent. This method can be efficient if unknown units are plotted against the curve that was produced with a sample of known concentration.
STORING OF ISOLATED RNA:
The storage step is the last step of RNA isolation. Storing RNA samples is very important. Also, keep it safe is very crucial. For storage, -80C is required. Always store isolated RNA in resuspended, single-use aliquots RNA storage containers.
THINGS TO CONSIDER DURING ISOLATION PROCEDURE OF RNA:
RNA isolation is a skill as well as an art. There are certain things that you need to keep in mind and apply to get the high quality of RNA. Here are some things to notice when isolating RNA:
- Wear gloves and make sure you change them frequently. Processes like this may fail if skin comes in contact and contamination happens.
- Always decontaminate pipettors, gel combs, and boxes.
- Always use reagents, tips, and tubes.
- Process tissue quickly. You can perform processing by disrupting in freezing, lysis buffer or storing in tissue.
- You need to grind tissue that is frozen in liquid nitrogen. Grind with a mortar or a pestle.
- Always clean your homogenizer in lysis buffer. Do this between samples in order to prevent contamination.
RNA isolation is a critical process for many biomedical techniques and researches. A high-quality RNA is the requirement of many biomedical technologies. To ensure that the RNA you extracted is pure and free of contamination, always take precautions and keep the lab environment uncontaminated. Other than that, RNA testing kit and DNA testing kits need to be free of contamination to provide high-quality results.
- RNA Extraction. Johnson, Mary. New Jersey, United States: The world of Laboratories, 2012.