The preparation of intact ribonucleic acid is difficult because of the action of
nucleases, which are liberated upon tissue homogenisation. In many cells, high
concentrations of the ribonucleases are reserved in the secretory granules and upon
disruption of the cell, they get mixed with the RNA and lead to its degradation.
Guanidinium chloride and thiocyanate are potent chaotropic agents that reduce
hydrophobic interactions and disrupt protein tertiary structures, disassociate proteinnucleic
acid complexes and disintegrate cellular structures. Guanidinium thiocyanate is
especially strong protein denaturant because both the cation and anion disrupt the
hydrophobic bonds between the amino acid side chains. RNA usually binds to proteins
within a cell and this agent disassociates the nucleoprotein complex, without disrupting
RNA structure. Thus RNA can be obtained by using these agents, after homogenisation
and low-speed centrifugation and precipitated with ethanol. The protocol below
explains the stepwise isolation of total RNA from cells and tissues using TRIzol
reagent which is the mono-phasic solution of phenol and guanidine thiocyanate.
Keywords: Centrifugation, CTAB, DEPC, Formaldehyde Gel, Guanidinium
chloride, Guanidine thiocyanate, Homogenization, Hydrophobic, Hot Phenol,
Nucleoprotein, Nucleic acid, Protein, phenol, RNA isolation, TRIzol.
