Troubleshooting Guide for Total RNA Extraction & Purification

RNA, also known as Ribonucleic acid, is the most important molecule responsible for storing and retaining genetic information. The major role of RNA is to convert the information stored into DNA into proteins. Ensure to buy reliable and good quality RNA isolation kits. One can use more than one method for RNA isolation. It does not matter which methodology one decides to use; the small traces of DNA always carry through.

There is significant discrepancy scientists encounter during the purification of RNA. Following are some of the problems, causes, and solutions:-

Clogged column

Homogenization is the process of making things uniform. In the quest of making things uniform, there is sometimes inadequacy of the sample. These things often lead to clogging of the column. It is advised to increase the time of homogenization. Using a larger DNA protection reagent volume also helps a lot for the total extraction and purification of RNA. The column used for the experimentation should not be overloaded. To ensure this, we should reduce the amount of starting material.

Low yield

One of the causes for a low yield can be the use of too many samples. For avoiding this, it is advised to store the input sample at -80 degree C before its use.

RNA degradation

One of the main reasons for the degradation of the RNA can be mishandling of the starting material. If the given sample is not protected by a preservation agent, Monarch DNA is often used as a reagent to maintain its integrity.

Low OD ratio

The most common method to quantitate DNA is using a spectrophotometer. It works on the premise that nucleic acid absorbs the ultraviolet light in a specific pattern. A photodetector is used to absorb the light. It is a general perception that the sample absorbs some light and the remaining light passes through. Nucleic acid and the light absorbed by the sample are directly proportional to each other. If more light is absorbed, the acid concentration increases. The combined effect of all the processes affects the OD ratio or optical density ratio.

Low OD ratio indicates the presence of residues in the sample.

To reduce this, one needs to make sure that the tip of the column is not in contact with the flow-through after the final wash. Before using the collection tubes again, remove the residual buffer.

DNA contamination

The cause of contamination or adulteration of DNA is that the genomic DNA is not removed by the column.

Some RNA isolation kits are available in the market for different price ranges depending on the specification. One needs to perform the on-column DNase I treatment to remove the genomic DNA. All these methods are being used for the troubleshooting and the purification of the RNA.

There are many RNA Extraction kits, such as Ambion cells, Trizol kits, etc. It is a very crucial step. For more than two decades, many scientists have initiated robust measures to improve products and services.

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