Abstract
Small RNAs, defined as noncoding 20–30-nt-long RNAs, are instrumental regulators of cellular processes in most eukaryotes. In this chapter we describe three methods for extracting small RNA from cells: a general method, one plant specific and a third particular to conifers. Further, protocols are given for the analysis and quantification of small RNAs using polyacrylamide gel-based approaches. A native streptavidin gel-shift assay, useful for measuring the relative amounts of multiple small RNAs simultaneously, is presented. To further characterize small RNAs biochemically, a sodium periodate assay probing for 2′, 3′ hydroxyl groups on the 3′ terminus of small RNAs is outlined.
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References
Hamilton AJ, Baulcombe DC (1999) A species of small antisense RNA in posttranscriptional gene silencing in plants. Science 286:950–952
Voinnet O (2009) Origin, biogenesis, and activity of plant microRNAs. Cell 136:669–687
Lagos-Quintana M, Rauhut R, Lendeckel W, Tuschl T (2001) Identification of novel genes coding for small expressed RNAs. Science 294: 853–858
Lau NC, Lim LP, Weinstein EG, Bartel DP (2001) An abundant class of tiny RNAs with probable regulatory roles in Caenorhabditis elegans. Science 294:858–862
Lee RC, Ambros V (2001) An extensive class of small RNAs in Caenorhabditis elegans. Science 294:862–864
Yu B, Yang Z, Li J, Minakhina S, Yang M, Padgett RW, Steward R et al (2005) Methylation as a crucial step in plant microRNA biogenesis. Science 307:932–935
Ebhardt HA, Thi EP, Wang MB, Unrau PJ (2005) Extensive 3′ modification of plant small RNAs is modulated by helper component-proteinase expression. Proc Natl Acad Sci USA 102:13398–13403
Yang Z, Ebright YW, Yu B, Chen X (2006) HEN1 recognizes 21–24 nt small RNA duplexes and deposits a methyl group onto the 2′ OH of the 3′ terminal nucleotide. Nucleic Acids Res 34:667–675
Rajagopalan R, Vaucheret H, Trejo J, Bartel DP (2006) A diverse and evolutionarily fluid set of microRNAs in Arabidopsis thaliana. Genes Dev 20:3407–3425
Morin RD, Aksay G, Dolgosheina E, Ebhardt HA, Magrini V, Mardis ER et al (2008) Comparative analysis of the small RNA transcriptomes of Pinus contorta and Oryza sativa. Genome Res 18:571–584
Dolgosheina EV, Morin RD, Aksay G, Sahinalp SC, Magrini V, Mardis ER et al (2008) Conifers have a unique small RNA silencing signature. RNA 14:1508–1515
Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
Kolosova N, Miller B, Ralph S, Ellis BE, Douglas C, Ritland K et al (2004) Isolation of high-quality RNA from gymnosperm and angiosperm trees. Biotechniques 36:821–824
Alwine JC, Kemp DJ, Stark GR (1977) Method for detection of specific RNAs in agarose gels by transfer to diazobenzyloxymethyl-paper and hybridization with DNA probes. Proc Natl Acad Sci USA 74:5350–5354
Ebhardt HA, Unrau PJ (2009) Characterizing multiple exogenous and endogenous small RNA populations in parallel with subfemtomolar sensitivity using a streptavidin gel-shift assay. RNA 15:724–731
Li Z, Brow DA (1993) A rapid assay for quantitative detection of specific RNAs. Nucleic Acids Res 21:4645–4646
Acknowledgments
Marie Curie International Incoming Fellow from the European Commission, Community Research and Development Information Service, Seventh Framework Programme (to H.A.E.). Operating grants from the Natural Sciences and Engineering Research Council of Canada and the Michael Smith Foundation (to P.J.U.).
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Ebhardt, H.A., Ovando, M.O., Unrau, P.J. (2012). Isolation and Biochemical Analysis of Plant Small RNAs. In: Watson, J., Wang, MB. (eds) Antiviral Resistance in Plants. Methods in Molecular Biology, vol 894. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-882-5_15
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DOI: https://doi.org/10.1007/978-1-61779-882-5_15
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