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Contaminants may also be identified in the same way as that for DNA by scanning

UV spectrophotometry; however, in the case of RNA a 260 nm : 280 nm ratio of

approximately 2 would be expected for a sample containing no protein (Section 5.8.1).

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 concen-

trations, 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 by further high salt solution. Finally,

the bound mRNA can be eluted using a low concentration of salt (Fig. 5.23). Nucleic

acid species may also be subfractionated by more physical means such as electro-

phoretic or chromatographic separations based on differences in nucleic acid fragment

sizes or physicochemical characteristics. Nanodrop spectrophotometer systems have

also aided the analysis of nucleic acids in recent years in allowing the full spectrum

of information whilst requiring only a very small (microlitre) sample volume.

Cellular mRNA (heterogeneous size transcripts)

AAAAAAA

AAAAAAA

AAAAAAA

Poly(dT) affinity column

AAAAA

TTTTT

TTTTTT

Poly(A)+ RNA binds to poly(dT)

AAAAAAA

TTTTT

TTTT

Non-poly(A)+ RNA and DNA are washed

through column in high salt concentrations

Poly(A) + RNA is eluted by changing

to low salt concentrations

TTTT

TTTTTT

AAAAAAA

TTTT

TTTT

Fig. 5.23Affinity chromatography of poly(A)þRNA.

167 5.7 Isolation and separation of nucleic acids