RNA (ribonucleic acid) is a molecule similar to DNA present in the cells for coding, decoding, regulation and expression of genes as protein synthesis and in some viruses act as their genetic material. RNAs are single stranded. RNA covert the message/code provided by the DNA into proteins for cellular activities (development, cellular differentiation, and changing environments) by translation.
RNA purification can be used for molecular analyses such as RT-PCR, cloning, in-vitro translation and transcription, transcriptome analysis, RNA structural and functional studies, depletion, and RNA sequencing sampling preparation. It is also essential for the development of diagnostics and drugs.
RNA extraction is the process of separating RNA from cells present in any biological sample. Mission is to separate pure RNA. The extracted RNA should be of good quality and in efficient amount. Quality should be determined by purity of RNA without contamination such as DNA, lipids and proteins. Avoiding things like detergents, temperature, bad handling, ribonuclease enzyme etc. which can denature RNA so should go through a certain protocol. RNA can be extracted by manual method as well as commercially available kits.
In RNA extraction and purification, Firstly disrupt the cells by adding guanidium thiocyanate and a reducing agent to the sample and then subject it to vigorous shaking or vortexing. This step will also break the disulphide bonds and inactivate the contaminant proteins present in the sample. After cell lysis, add phenol and chloroform-isoamyl alcohol to separate RNA sample from the solution. The aqueous phase contains RNA which is transferred to a separate tube, add isopropanol and centrifuge the solution to precipitate the RNA. Washing the precipitate with 75% ethanol will then remove any impurities from the sample.