RNA mapping protocols: Northern blot and amplification of cDNA ends

Abstract

Mapping of transcribed mRNA and protein coding sequences is the initial step in functional characterization of genomic sequences. There are several techniques to identify transcribed sequences; however, Northern blot analysis remains a standard method for detection and quantification of mRNA levels despite the relatively recent availability of other methods, such as RT-PCR (reverse transcriptase polymerase chain reaction), which are also sensitive, accurate, cost-effective, and simple to use. Northern blot analysis provides a direct relative comparison of message abundance between samples on a single membrane. It is also the preferred method for determining transcript size and detecting alternatively spliced isoforms. Other methods utilizing RT-PCR allow detection and cloning of isolated complementary DNA (cDNA) domains and ends from cellular RNA or commercially available cDNA libraries.