Isolation of RNA

The methods used for RNA isolation are very similar to those described for DNA; however, RNA molecules are relatively short and therefore less easily damaged by shearing, so cell disruption can be more vigorous. RNA is, however, very vulnerable to digestion by RNases.
which are present endogenously in various concentrations in certain cell types and exogenously on fingers. Gloves should therefore be worn and a strong detergent should be included in the isolation medium to denature immediately any RNases. Subsequent deproteinisation should be particularly rigorous, since RNA is often tightly associated with proteins.

DNase treatment can be used to remove DNA and RNA can be precipitated by ethanol. One reagent in which is commonly used in RNA extraction is guanadinium thiocyanate, which is both a strong inhibitor of RNase and a protein denaturant. It is possible to check the integrity of an RNA extract by analysing it by agarose gel electrophoresis.

The most abundant RNA species are rRNA molecules, 23S and 16S for prokaryotes and 18S and 28S for eukaryotes. These appear as discrete bands on the agarose gel and indicate that the other RNA components are likely to be intact. This is usually carried out under denaturing conditions to prevent secondary structure formation in the
RNA. The concentration of the RNA may be estimated by using UV spectrophotometry. At 260 nm, 1 absorbance unit equates to 40 μgml^-1 of RNA and therefore :
40 *  A₂₆₀ = concentration of RNA sample (μgml^-1)
Contaminants may also be identified in the same way as for DNA by scanning UV spectrophotometry; however, in the case of RNA a 260 nm:280nm ratio of approximately 2 would be expected for a sample containing no contamination.
In many cases, it is desirable to isolate eukaryotic mRNA, which constitutes only 2–5% of cellular RNA, from a mixture of total RNA molecules. This may be carried out by affinity chromatography on oligo (dT)-cellulose columns. At high salt concentrations, the mRNA containing poly(A) tails binds to the complementary oligo(dT) molecules of
the affinity column and so mRNA will be retained; all other RNA molecules can be washed through the column with further high-salt solution. Finally, the bound mRNA can be eluted using a low concentration of salt. Nucleic acid species may also be subfractionated by more physical means such as electrophoretic or chromatographic separations based on differences in nucleic acid fragment sizes or physicochemical characteristics.