Objective:

 To understand isolation of RNA, the most critical step in performing most of the molecular biology experiments.

Principle:

RNA (Ribonucleic acid) is a polymeric substance present in living cells and many viruses, consisting of a long single-stranded chain of phosphate and ribose units with the  nitrogen bases adenine, guanine, cytosine, and uracil, which are bonded to the ribose sugar. RNA is used in all the steps of protein synthesis in all living cells and carries the genetic information for many viruses.

The isolation of RNA with high quality is a crucial step required to perform various molecular biology experiment. TRIzol Reagent is a ready-to-use reagent used for RNA isolation from cells and tissues.TRIzol works by maintaining RNA integrity during tissue homogenization, while at the same time disrupting and breaking down cells and cell components. Addition  of  chloroform, after the centrifugation, separates the solution  into  aqueous and organic phases. RNA  remains only in  the  aqueous  phase.

After transferring the aqueous phase, RNA can be recovered by precipitation with isopropyl alcohol. But the DNA and proteins can recover by sequential separation after the removal of aqueous phase. Precipitation with ethanol requires DNA from the interphase, and an additional precipitation with isopropyl alcohol requires proteins from the organic phase. Total RNA extracted by TRIzol Reagent is free from the contamination of protein and DNA. This RNA can be used in Northern blot analysis, in vitro translation, poly (A) selection, RNase protection  assay, and molecular cloning.