Methods in Molecular Biology Volume 847, 2012, pp 25-32

Use of Northern Blotting for Specific Detection of Small RNA Molecules in Transgenic Plants

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Abstract

Small RNAs (20–24 nucleotides long and nonprotein coding) have been increasingly investigated. They are responsible for phenomena described as RNA interference (RNAi), cosuppression, gene silencing, or quelling. Major classes of small RNAs include microRNAs (miRNAs) and small interfering RNAs (siRNAs), which differ in their biosynthesis. MiRNAs control the expression of cognate target genes by binding to reverse complementary sequences, resulting in cleavage or translational inhibition of the target RNA. SiRNAs have similar structure, function, and biogenesis as miRNAs; siRNAs derive from long double-stranded RNA of transgenes, endogenous repeat sequences, or transposons. Understanding these fundamental processes requires the sensitive and specific detection of small RNA species. In this report, we present a simple Northern blot protocol for small RNAs in transgenic plants.