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A number of protocols are available for the analysis of RNA transcripts: Northern analysis (
see
Chapter 18 ), S1 mapping, primer extension, RNase AT
1
mapping (
see
Chapter 20 ), and the more recent reverse transcriptase/
PCR
amplification (
see
Chapters 22 and24 ). These protocols have been applied to the analysis of plant RNAs and all vary in the degree of sensitivity of detection and the information generated. Primer extension assays have been used mainly in mapping of 5′ termini of RNA transcripts, but have also been of value, in, for example, analysis of nuclear pre-mRNA splicing (
1
–
3
), and in the detection of low abundance RNA species (
2
). There are four steps involved in performing a primer extension assay. First, selection and preparation of a labeled primer complementary to the RNA transcript of interest. Second, hybridization of the primer complementary to a region of the RNA under study. Third, extension from the primer that is catalyzed by an RNA-dependent
DNA
polymerase (reverse transcriptase) using RNA as the template to synthesize a
cDNA
strand. Fourth, analysis of the extended cDNA products on denaturing polyacrylaminde gels and autoradiography.
