Primer Extension Assay

A number of protocols are available for the analysis of RNA transcripts: Northern analysis (see Chapter 18 ), S1 mapping, primer extension, RNase AT₁ 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.