Profiling of short RNAs using Helicos single-molecule sequencing.

The importance of short (<200 nt) RNAs in cell biogenesis has been well documented. These short RNAs include crucial classes of molecules such as transfer RNAs, small nuclear RNA, microRNAs, and many others (reviewed in Storz et al., Annu Rev Biochem 74:199-217, 2005; Ghildiyal and Zamore, Nat Rev Genet 10:94-108, 2009). Furthermore, the realm of functional RNAs that fall within this size range is growing to include less well-characterized RNAs such as short RNAs found at the promoters and 3' termini of genes (Affymetrix ENCODE Transcriptome Project et al., Nature 457:1028-1032, 2009; Davis and Ares, Proc Natl Acad Sci USA 103:3262-3267, 2006; Kapranov et al., Science 316:1484-1488, 2007; Taft et al., Nat Genet 41:572-578, 2009; Kapranov et al., Nature 466:642-646, 2010), short RNAs involved in paramutation (Rassoulzadegan et al., Nature 441:469-474, 2006), and others (reviewed in Kawaji and Hayashizaki, PLoS Genet 4:e22, 2008). Discovery and accurate quantification of these RNA molecules, less than 200 bases in size, is thus an important and also challenging aspect of understanding the full repertoire of cellular and extracellular RNAs. Here, we describe the strategies and procedures we developed to profile short RNA species using single-molecule sequencing (SMS) and the advantages SMS offers.