Detection of small non-coding RNAs.

Abstract

Gene expression is regulated at several levels in plants, and one of the most recently discovered regulatory layers involve short RNAs. Short RNAs are produced through several pathways and target either mRNAs or genomic DNA. Different classes of short RNAs have slightly different sizes and detection of their accumulation is an important step in validating and studying non-coding short RNAs. Northern blotting is routinely used to detect short RNAs because it gives information about both the amount and size of the analysed short RNAs. Choice of the right RNA extraction protocol is crucial when short RNAs are being studied, because several routinely used commercial RNA extraction kits do not yield any short RNAs. This chapter describes optimised RNA extraction methods, which give good yields of short RNAs, and separation, transfer and hybridisation protocols to study the accumulation of short RNAs.