RNA Mapping Protocols: Northern Blot and Amplification of cDNA Ends
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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.