Abstract
Small, noncoding RNAs have proven to be powerful and ubiquitous agents of gene regulation in eukaryotic genomes. Since their initial discovery, several methods have been developed for directly cloning and sequencing these tiny RNA species. These cloning methods are presented along with a discussion of ways to enhance cloning efficiency and success. In addition, major advances in massively parallel, next-generation sequencing methods are presented as they impact small RNA studies. Finally, results of the application of one of the extant in vitro cloning methods to a marsupial mammal model are presented.
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References
Napoli C, Lemieux C, Jorgensen R Introduction of a chimeric chalcone synthase gene into petunia results in reversible co-suppression of homologous genes in trans. Plant Cell 1990; 2: 279–89.
van der Krol AR, Mur LA, Beld M, et al. Flavinoid genes in petunia: addition of a limited number of gene copies may lead to a suppression of gene expression. Plant Cell 1990; 2: 291–9.
Romano N, Macino G Quelling: transient inactivation of gene expression in Neurospora crassa by transformation with homologous sequences. Molecular Microbiology 1992; 6: 3343–53.
Cogoni , Macino G Isolation of quelling-defective (qde) mutants impaired in posttranscriptional transgene-induced gene silencing in Neurospora crassa. Proc Natl Acad Sci USA 1997; 94: 10233–8.
Fire A, Xu S, Montgomery MK, et al. Potent and specific genetic interference by double stranded RNA in Caenorhabditis elgans. Nature 1998; 391: 806–11.
Meister G, Tuschl T Mechanisms of gene silencing by double stranded RNA. Nature 2004; 431: 343–9.
Lee RC, Feinbaum RL, Ambros V The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell 1993; 75: 843–54.
Wightman B, Ha I, Ruvkun G Posttranscriptional regulation of the heterochronic gene lin-14 by lin-4 mediates temporal pattern-formation in C. elegans. Cell 1993; 75: 855–62.
Lagos-Quintana M, Rauhut R, Lendeckel W, Tuschl T Identification of novel genes coding for small expressed RNAs. Science 2001; 294: 853–8.
Lau NC, Lim LP, Weinstein EG, Bartel DP An abundant class of tiny RNAs with probable regulatory roles in Caenorhabditis elegans. Science 2001; 294: 858–62.
Lee R, Ambros V An extensive class of small RNAs in Caenorhabditis elegans. Science 2001; 294: 862–4.
Reinhart BJ, Weinstein EG, Rhoades M, Bartel B, Bartel DP MicroRNAs in plants. Genes and Development 2002; 16: 1616–26.
Du T, Zamore PD MicroPrimer: the biogenesis and function of microRNA. Development 2005; 132: 4645–52.
Kim DH, Rossi JJ Strategies for silencing human disease using RNA interference. Nat Rev Genet. 2007: 8: 173–84.
Agaard L, Rossi JJ RNAi therapeutics: principles, prospects and challenges. Adv Drug Deliv Rev. 2007; 59: 75–86.
Kim VN Small RNAs: Classification, biogenesis, and function. Mol Cells 2005; 19:1–15.
Tissot C Analysis if miRNA contant in total RNA preparations using the Agilent 2100 bioanalyzer. Agilent Technologies Publication 5989–7870EN.
Berezikov E, Cuppen E, Plasterk RHA Approaches to microRNA discovery. Nature Genetics 2006; 38: S2–S7.
Fu H, Tie Y, Xu C, et al. Identification of human fetal liver miRNAs by a novel method. FEBS Lett 2005; 579: 3849–54.
Pfeffer S, Lagos-Quintana M, Tuschl T Cloning of small RNA molecules. In: Ausubel FM, Brent R, Kingston RE, et al., eds. Current Protocols in Molecular Biology, Vol. 4. 2003: 26.4.1–26.4.18.
Cummins JM, He Y, Leary RJ, et al. The colorectal microRNAome. Proc. Natl Acad Sci USA 2006; 103: 3687–92.
Lau NC, Lim LP, Weinstein EG, Bartel DP An abundant class of tiny RNAs with probable regulatory roles in Caenorhabditis elegans. Science 2001; 294: 858-62.
Pfeffer S, Sewer A, Lagos-Quintana M, et al. Identification of microRNAs of the herpesvirus family. Nature Methods 2005; 2: 269–76.
Aravin A, Tuschl T Identification and characterization of small RNAs involved in RNA silencing. FEBS Lett 2005; 579: 5830–40.
Chen PY, Manninga H, Slanchev K, et al. The developmental miRNA profiles of zebrafish as determined by small RNA cloning. Genes Devel 2005; 19: 1288–93.
Velculescu VE, Zhang L, Vogelstein B, Kinzler KW Serial analysis of gene expression. Science 1995; 270: 484–7.
Pak J, Fire A Distinct populations of primary and secondary effectors during RNAi in C. elegans. Science 2007; 315: 241–4.
Devor EJ, Huang L, Abdukarimov A, Abdurakhmonov IY Methodologies for in vitro cloning of small RNAs and application for plant genome(s). Intl J Plant Genomics 2009; ID 915061.
Griffiths-Jones S miRBase: the microRNA sequence database. Methods Mol Biol. 2006; 342: 129–38.
Griffiths-Jones S, Saini HK, Dongen SV, Enright AJ miRBase: tools for microRNA genomics. Nucleic Acids Res 2008; 36 (Database Issue): D154–D158.
Ambros V, A uniform system for microRNA annotation. RNA 2003; 9: 277–9.
Margulies M, Egholm M, Altman WE Genome sequencing in microfabricated high-density picolitre reactors. Nature 2005; 437: 376–80.
Mardis E The impact of next-generation sequencing technology on genetics. Trends in Genetics 2008; 24: 133–41.
Parameswaran P, Jalili R, Tao L, et al. A pyrosequencing-tailored nucleotide barcode design unveils opportunities for large-scale sample multiplexing. Nucleic Acids Res. 2007; 35: e130.
Hamady M, Walker JJ, Harris JK, et al., Error-correcting barcoded primers for pyrosequencing hundreds of samples in multiplex. Nature Methods 2008; 5: 235–7.
Ruby JG, Jan C, Palyer C et al., Large-scale sequencing reveals 21U-RNAs and additional microRNAs and endogenous siRNAs in C. elegans. Cell 2006; 127: 1193–207.
Hutchinson CA DNA sequencing: bench to bedside and beyond. Nucleic Acids Res 2007; 35: 6227–37.
Shendure J, Porreca GJ, Reppas NB, et al. Accurate multiplex polony sequencing of an evolved bacterial genome. Science 2005; 309: 1728–32.
Mikkelsen TS, Wakefield MJ, Aken B, et al., Genome of the marsupial Monodelphis domestica reveals innovation in non-coding sequences. Nature 2007; 447: 167–77.
Devor EJ, Samollow PB. In vitro and in silico annotation of conserved and non-conserved microRNAs in the genome of the marsupial Monodelphis domestica. J Hered 2008; 99: 66–72.
Warren WC, Hillier LW, Marshall Graves, JA Genome analysis of the platypus reveals unique signatures of evolution. Nature 2008; 453: 175–83.
Devor EJ, Huang L, Samollow PB piRNA-like RNAs in the marsupial Monodelphis domestica identify transcription clusters and likely marsupial transposon targets. Mammalian Genome 2008; 19: 581–586.
O’Donnell KA, Boeke JD Mighty Piwis defend the germline against genome intruders. Cell 2007; 129: 37–44.
Kim VN Small RNAs just got bigger, Piwi-interacting RNAs (piRNAs) in mammalian testes. Genes and Development 2006; 20: 1993–7.
Brennecke J, Aravin AA, Stark A, et al., Discrete small RNA-generating loci as master regulators of transposon activity in Drosophila. Cell 2007; 128: 1–15.
Aravin AA, Hannon GJ, Brennecke J The Piwi-piRNA pathway provides an adaptive defense in the transposon arms race. Science 2007; 318: 761–4.
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Devor, E.J., Huang, L. (2011). Cloning Small RNAs. In: Harper, S. (eds) RNA Interference Techniques. Neuromethods, vol 58. Humana Press. https://doi.org/10.1007/978-1-61779-114-7_5
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DOI: https://doi.org/10.1007/978-1-61779-114-7_5
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