RNA Extraction.

RNases enzyme is one of the main problem of the RNA extraction. that degraded the RNA because RNases is very stable, found every everywhere, active enzyme that do not required any co-factors and maintain activity even during the autoclaving.

Avoiding RNase Contamination

• RNase inhibitors are added to the extraction buffer to inhibit the endogenous RNase: Guanidine salt, protein based RNase inhibitors (RNain)
• To remove exogenous RNase Contamination: DEPC (Diethyl pyrocarbonate)
• All reagents are inactive the RNases by denature the proteins.

RNA Extraction Methods

Guanidinium thiocyanate phenol-chloroform extraction method: Guanidinium thiocyanate–phenol solution (TRI reagent) – disrupts the cells, denature the proteins and deactivate the nucleases. Under acidic conditions (pH 4-6) DNA and proteins distributed into the phenol : chloroform phase and the RNA remains in the aqueous phase. RNA is recovered by precipitation by isopropanol.

High-salt lithium chloride method: cell breakage in low pH, high salt buffer in the presence of
RNase inhibitors. Protein and DNA are removed by acidic phenol:choloroform extraction
and RNA is recovered by lithium chloride precipitation

Solid phase extraction: In presence of a high concentration of salts RNA will bind to silica surface, protein will not binds. then nucleic acids are eluted under the low or non – salt conditions.

References