title: FDF-PAGE: a powerful technique revealing previously undetected small RNAs sequestered by complementary transcripts.
abstract: Small RNAs, between 18nt and 30nt in length, are a diverse class of non-coding RNAs that mediate a range of cellular processes, from gene regulation to pathogen defense. They guide ribonucleoprotein complexes to their target nucleic acids by Watson-Crick base pairing. We report here that current techniques for small RNA detection and library generation are biased by formation of RNA duplexes. To address this problem, we established FDF-PAGE (fully-denaturing formaldehyde polyacrylamide gel electrophoresis) to prevent annealing of sRNAs to their complement. By applying FDF-PAGE, we provide evidence that both strands of viral small RNA are present in near equimolar ratios, indicating that the predominant precursor is a long double-stranded RNA. Comparing non-denaturing conditions to FDF-PAGE uncovered extensive sequestration of miRNAs in model organisms and allowed us to identify candidate small RNAs under the control of competing endogenous RNAs (ceRNAs). By revealing the full repertoire of small RNAs, we can begin to create a better understanding of small RNA mediated interactions.
authors.affiliation: Plant Sciences Department, Cambridge University, Cambridge, CB2 3EA, UK.
authors.forename: C Jake
authors.identifier: http://orcid.org/0000-0001-5120-0377
authors.initials: CJ
authors.lastname: Harris
authors.affiliation: Plant Sciences Department, Cambridge University, Cambridge, CB2 3EA, UK School of Biological Sciences, Edinburgh University, Edinburgh, EH9 3JH, UK.
authors.forename: Attila
authors.initials: A
authors.lastname: Molnar
authors.affiliation: Plant Sciences Department, Cambridge University, Cambridge, CB2 3EA, UK.
authors.forename: Sebastian Y
authors.initials: SY
authors.lastname: Müller
authors.affiliation: Plant Sciences Department, Cambridge University, Cambridge, CB2 3EA, UK dcb40@cam.ac.uk.
authors.forename: David C
authors.initials: DC
authors.lastname: Baulcombe
mesh_terms: D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D005557:Formaldehyde; D016679:Genome, Viral; D009691:Nucleic Acid Denaturation; D058727:RNA, Small Untranslated / Q000032:analysis* / Q000737:chemistry; D012367:RNA, Viral / Q000032:analysis / Q000235:genetics; D017423:Sequence Analysis, RNA; D014026:Nicotiana / Q000235:genetics
chemical_list: D058727:RNA, Small Untranslated; D012367:RNA, Viral; D005557:Formaldehyde