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Plant Epigenetics pp 127–139Cite as

Analysis of Small RNA Populations Using Hybridization to DNA Tiling Arrays

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 1456))

Abstract

Epigenetic response to stress in plants involves changes in DNA methylation, histone modifications, and expression of small noncoding RNAs (sRNA). Here we present the method of analysis of differential expression of sRNA populations using DNA tiling arrays. sRNA extracted from Arabidopsis thaliana plants exposed to pathogen elicitor or control plants were reverse-transcribed into cDNAs, and subsequently hybridized after labeling to a custom-made DNA tiling array covering Arabidopsis chromosome 4. We first designed a control experiment with eight cDNA clones corresponding to sequences located on chromosome 4 and obtained robust and specific hybridization signals. Furthermore, hybridization signals along chromosome 4 were in good agreement with sRNA abundance as previously determined by massive parallel sequence signature (MPSS) in the case of untreated plants, but differed substantially after stress treatment. These results demonstrate the utility of hybridization to DNA tiling arrays to detect major changes in sRNA abundance.

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References

  1. Baulcombe D (2004) RNA silencing in plants. Nature 431:356–363

    Article  CAS  PubMed  Google Scholar 

  2. Brodersen P, Voinnet O (2006) The diversity of RNA silencing pathways in plants. Trends Genet 22:268–280

    Article  CAS  PubMed  Google Scholar 

  3. Baev V, Milev I, Naydenov M, Vachev T, Apostolova E, Mehterov N, Gozmanva M, Minkov G, Sablok G, Yahubyan G (2014) Insight into small RNA abundance and expression in high- and low-temperature stress response using deep sequencing in Arabidopsis. Plant Physiol Biochem 84:105–114

    Article  CAS  PubMed  Google Scholar 

  4. Sunkar R, Zhu JK (2004) Novel and stress-regulated MicroRNAs and other small RNAs from Arabidopsis. Plant Cell 16:2001–2019

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Navarro L, Dunoyer P, Jay F, Arnold B, Dharmasiri N, Estelle M, Voinnet O, Jones JD (2006) A plant miRNA contributes to antibacterial resistance by repressing auxin signaling. Science 312:436–439

    Article  CAS  PubMed  Google Scholar 

  6. Sunkar R, Chinnusamy V, Zhu J, Zhu JK (2007) Small RNAs as big players in plant abiotic stress responses and nutrient deprivation. Trends Plant Sci 12:301–309

    Article  CAS  PubMed  Google Scholar 

  7. Bilichak A, Ilnytskyy Y, Wóycicki R, Kepeshchuk N, Fogen D, Kovalchuk I (2015) The elucidation of stress memory inheritance in Brassica rapa plants. Front Plant Sci 6:5

    PubMed  PubMed Central  Google Scholar 

  8. Greenberg JT, Yao N (2004) The role and regulation of programmed cell death in plant-pathogen interactions. Cell Microbiol 6:201–211

    Article  CAS  PubMed  Google Scholar 

  9. Wei ZM, Laby RJ, Zumoff CH, Bauer DW, He SY, Collmer A, Beer SV (1992) Harpin, elicitor of the hypersensitive response produced by the plant pathogen Erwinia amylovora. Science 257:85–88

    Article  CAS  PubMed  Google Scholar 

  10. Llave C, Kasschau KD, Rector MA, Carrington JC (2002) Endogenous and silencing associated small RNAs in plants. Plant Cell 14:1605–1619

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Pfeffer S, Lagos-Quintana M, Tuschl T (2005) Cloning of small RNA molecules. Curr Protoc Mol Biol 26(Unit 26.4):26410–26418

    Google Scholar 

  12. Hafner M, Landgraf P, Ludwig J, Rice A, Ojo T, Lin C, Holoch D, Lim C, Tuschl T (2008) Identification of microRNAs and other small regulatory RNAs using cDNA library sequencing. Methods 44:3–12

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Martienssen RA, Doerge RW, Colot V (2005) Epigenomic mapping in Arabidopsis using tiling microarrays. Chromosome Res 13:299–308

    Article  CAS  PubMed  Google Scholar 

  14. Vaughn MW, Tanurd IM, Lippman Z, Jiang H, Carrasquillo R, Rabinowicz PD, Dedhia N, McCombie WR, Agier N, Bulski A, Colot V, Doerge RW, Martienssen RA (2007) Epigenetic Natural Variation in Arabidopsis thaliana. PLoS Biol 5, e174

    Article  PubMed  PubMed Central  Google Scholar 

  15. Lu C, Tej SS, Luo S, Haudenschild CD, Meyers BC, Green PJ (2006) Elucidation of the small RNA component of the transcriptome. Science 309:1567–1569

    Article  Google Scholar 

  16. Nakano M, Nobuta K, Vemaraju K, Tej SS, Skogen JW, Meyers BC (2006) Plant MPSS databases: signature-based transcriptional resources for analyses of mRNA and small RNA. Nucleic Acids Res 34:D731–D735

    Article  CAS  PubMed  Google Scholar 

  17. Thibaud-Nissen F, Wu H, Richmond T, Redman JC, Johnson C, Green R, Arias J, Town CD (2006) Development of Arabidopsis whole-genome microarrays and their application to the discovery of binding sites for the TGA2 transcription factor in salicylic acid-treated plants. Plant J 47:152–162

    Article  CAS  PubMed  Google Scholar 

  18. Sorefan K, Pais H, Hall AE, Kozomara A, Griffiths-Jones S, Moulton V, Dalmay T (2012) Reducing ligation bias of small RNAs in libraries for next generation sequencing. Silence 3:4

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Fuchs RT, Sun Z, Zhuang F, Robb GB (2015) Bias in Ligation-Based Small RNA Sequencing Library Construction Is Determined by Adaptor and RNA Structure. PLoS One 10(5), e0126049

    Article  PubMed  PubMed Central  Google Scholar 

  20. Boccara M, Sarazin A, Billoud B, Jolly V, Martienssen R, Baulcombe D, Colot V (2007) New approaches for the analysis of Arabidopsis thaliana small RNAs. Biochimie 89:1252–1256

    Article  CAS  PubMed  Google Scholar 

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Acknowledgments

MB was supported by a Visiting Scientist Fellowship from INRA. VC and DB are members of the European Union Network of Excellence “The Epigenome.”

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Authors and Affiliations

  1. PSL Research University, Institut de Biologie de l’Ecole Normale Supérieure (IBENS), CNRS UMR 8197, INSERM U1024, Ecole NormaleSupérieure, 46 rue d’Ulm, Paris, F75005, France

    Martine Boccara, Alexis Sarazin, Agnes Bulski & Vincent Colot

  2. Atelier de bioinformatique, Sorbonne Universités, UMR 7205 (MNHN, UPMC, CNRS, EPHE), Museum national d’histoire naturelle, rueBuffon, Paris, F-7523, France

    Bernard Billoud

  3. The Sainsbury Laboratory, John Innes Centre, Norwich, UK

    Louise Chapell & David Baulcombe

Authors
  1. Martine Boccara
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  2. Alexis Sarazin
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  3. Bernard Billoud
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  4. Agnes Bulski
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  5. Louise Chapell
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  6. David Baulcombe
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  7. Vincent Colot
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Corresponding author

Correspondence to Martine Boccara .

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Editors and Affiliations

  1. Department of Biological Sciences, University of Lethbridge, Lethbridge, Alberta, Canada

    Igor Kovalchuk

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Boccara, M. et al. (2017). Analysis of Small RNA Populations Using Hybridization to DNA Tiling Arrays. In: Kovalchuk, I. (eds) Plant Epigenetics. Methods in Molecular Biology, vol 1456. Humana Press, Boston, MA. https://doi.org/10.1007/978-1-4899-7708-3_11

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  • DOI: https://doi.org/10.1007/978-1-4899-7708-3_11

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  • Publisher Name: Humana Press, Boston, MA

  • Print ISBN: 978-1-4899-7706-9

  • Online ISBN: 978-1-4899-7708-3

  • eBook Packages: Springer Protocols

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