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Purification of Viral RNA through Phenol-Chloroform

Viral analysis of biological and environmental samples, such as animals, insects, plants, fungi, and bacteria is an important process in molecular biology. It aims to identify significant viruses from samples and discover a wider variety of diseases. To ensure optimum performance and accurate result in the assays, scientists first need to purify viral RNA samples.

Purification of RNA involves separating the RNA virus from the sample using reliable molecular technologies. There are several methods of purifying RNA, the most widely used technique is the phenol-chloroform purification.

Phenol-chloroform is a liquid to liquid purification technique in molecular biology. During the process, scientists mix equal volume of phenol-chloroform and an aqueous sample to form a biphasic mixture, a property of liquid that allows mixture to proportionately combine. The sample is then separated by phase through centrifugation or the use of centrifugal energy. Using a centrifuge, the sample is placed on a tube and is spun at a high speed causing the heavier components to submerge on the bottom while the lighter components are layered on top. The scientists recover the viral RNA from the aqueous phase by precipitation with 2-propanol or ethanol.

The process of viral RNA purification can be complicated. Scientists basically take extra care in handling samples and performing the process because of the presence of ribonuclease enzymes in tissues, which may rapidly degrade the RNA.

The column based purification method, such as the purification by silica is an alternative method to purify viral RNA. Unlike phenol-chloroform purification, this method is based on solid phase.  Depending on the pH level of nucleic acids, RNA may bind with the solid phase silica to separate from the sample. Column based purification method is faster than phenol-chloroform purification; however for a higher rate of purification, scientists would usually use phenol-chloroform purification method.

Additional resources:

http://en.wikipedia.org/wiki/RNA_extraction

http://en.wikipedia.org/wiki/Guanidinium_thiocyanate-phenol-chloroform_extraction

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