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detection of particular RNA transcripts by blotting extracted mRNA and immobilising it

to a nylon membrane (Section 5.9.2). Subsequent hybridisation with labelled gene probes

allows precise determination of the size and nature of a transcript. However, much use

has been made of a number of nucleases that digest only single-stranded nucleic acids

and not double-stranded molecules. In particular theribonuclease protection assay

(RPA) has allowed much information to be gained regarding the nature of mRNA

transcripts (Fig. 6.38). In the RPA single-stranded mRNA is hybridised in solution to a

labelled single-stranded RNA probe which is in excess. The hybridised part of the

complex becomes protected whereas the unhybridised part of the probe made from

RNA is digested with RNase A and RNase T1. The protected fragment may then be

analysed on a high-resolution polyacrylamide gel. This method may give valuable

information regarding the mRNA in terms of the precise structure of the transcript

(transcription start site, intron/exon junctions, etc.). It is also quantitative and requires

less RNA than aNorthern blot. A related technique,S1 nuclease mapping,issimilar

although the unhybridised part of a DNA probe, rather than an RNA probe, is digested,

this time with the enzyme S1 nuclease.

The PCR has also had an impact on the analysis of RNA via the development of a

technique known asreverse transcriptase–PCR(RT–PCR). Here the RNA is isolated

and a first strand cDNA synthesis undertaken with reverse transcriptase; the cDNA is

then used in a conventional PCR (Section 6.2.5). Under certain circumstances a

number of thermostable DNA polymerases have reverse transcriptase activity which

obviates the need to separate the two reactions and allows the RT–PCR to be carried

out in one tube. One of the main benefits of RT–PCR is the ability to identify rare or

low levels of mRNA transcripts with great sensitivity. This is especially useful when

Total RNA isolation

Specific mRNA
Labelled RNA probe
Markers RNA/Probe

Hybridisation of probe

and specific RNA

RNase digestion of

unhybridised RNA

RNA purification

and PAGE analysis

Fig. 6.38Steps involved in the ribonuclease protection assay (RPA). PAGE, polyacrylamide gel electrophoresis.

241 6.8 Analysing genes and gene expression