Analysis of Small RNA Populations Using Hybridization to DNA Tiling Arrays

  • Martine BoccaraEmail author
  • Alexis Sarazin
  • Bernard Billoud
  • Agnes Bulski
  • Louise Chapell
  • David Baulcombe
  • Vincent Colot
Part of the Methods in Molecular Biology™ book series (MIMB, volume 631)


Small RNA (sRNA) populations extracted from Arabidopsis plants submitted or not to biotic stress, were reverse-transcribed into cDNAs, and these were subsequently hybridized after labelling 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 small RNA populations.

Key words

Small RNA cDNA libraries cy-dye indirect labelling Hypersensitive response Microarray Harpin 



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


  1. 1.
    Baulcombe D (2004) RNA silencing in plants. Nature 431:356–363CrossRefPubMedGoogle Scholar
  2. 2.
    Brodersen P, Voinnet O (2006) The diversity of RNA silencing pathways in plants. Trends Genet 22:268–280CrossRefPubMedGoogle Scholar
  3. 3.
    Jones-Rhoades MW, Bartel DP (2004) Compu­tational identification of plant microRNAs and their targets including a stress-induced miRNA. Mol Cell 14:787–799CrossRefPubMedGoogle Scholar
  4. 4.
    Sunkar R, Zhu JK (2004) Novel and stress-regulated MicroRNAs and other small RNAs from Arabidopsis. Plant Cell 16:2001–2019CrossRefPubMedGoogle Scholar
  5. 5.
    Navarro L, Dunoyer P, Jay F, Arnold B, Dharmasiri N, Estelle M et al (2006) A plant miRNA contributes to antibacterial resistance by repressing auxin signaling. Science 312:436–439CrossRefPubMedGoogle Scholar
  6. 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 12:301–309CrossRefPubMedGoogle Scholar
  7. 7.
    Greenberg JT, Yao N (2004) The role and regulation of programmed cell death in plant-pathogen interactions. Cell Microbiol 6:201–211CrossRefPubMedGoogle Scholar
  8. 8.
    Wei ZM, Laby RJ, Zumoff CH, Bauer DW, He SY, Collmer A et al (1992) Harpin, elicitor of the hypersensitive response produced by the plant pathogen Erwinia amylovora. Science 257:85–88CrossRefPubMedGoogle Scholar
  9. 9.
    Llave C, Kasschau KD, Rector MA, Carrington JC (2002) Endogenous and silencing associated small RNAs in plants. Plant Cell 14:1605–1619CrossRefPubMedGoogle Scholar
  10. 10.
    Pfeffer S, Lagos-Quintana M, Tuschl T (2005) Cloning of small RNA molecules. Curr Protoc Mol Biol 26, Unit 26.4:26410–26418Google Scholar
  11. 11.
    Hafner M, Landgraf P, Ludwig J, Rice A, Ojo T, Lin C et al (2008) Identification of microRNAs and other small regulatory RNAs using cDNA library sequencing. Methods 44:3–12CrossRefPubMedGoogle Scholar
  12. 12.
    Martienssen RA, Doerge RW, Colot V (2005) Epigenomic mapping in Arabidopsis using tiling microarrays. Chromosome Res 13:299–308CrossRefPubMedGoogle Scholar
  13. 13.
    Vaughn MW, Tanurdzic´ M, Lippman Z, Jiang H, Carrasquillo R, Rabinowicz PD et al (2007) Epigenetic natural variation in Arabidopsis thaliana. PLoS Biol 5:e174CrossRefPubMedGoogle Scholar
  14. 14.
    Lu C, Tej SS, Luo S, Haudenschild CD, Meyers BC, Green PJ (2006) Elucidation of the small RNA component of the transcriptome. Science 309:1567–1569CrossRefGoogle Scholar
  15. 15.
    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–D735CrossRefPubMedGoogle Scholar
  16. 16.
    Thibaud-Nissen F, Wu H, Richmond T, Redman JC, Johnson C, Green R et al (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 47:152–162CrossRefPubMedGoogle Scholar
  17. 17.
    Boccara M, Sarazin A, Billoud B, Jolly V, Martienssen R, Baulcombe D et al (2007) New approaches for the analysis of Arabidopsis thaliana small RNAs. Biochimie 89:1252–1256CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Martine Boccara
    • 1
    Email author
  • Alexis Sarazin
    • 2
  • Bernard Billoud
    • 3
  • Agnes Bulski
    • 4
  • Louise Chapell
    • 5
  • David Baulcombe
    • 5
  • Vincent Colot
    • 1
  1. 1.Unité de Recherche en Génomique Végétale (URGV), INRA/CNRS/UEVEEvry cedexFrance
  2. 2.CNRS UMR8186, Département de biologieEcole Normale SupérieureParis cedex 05France
  3. 3.Atelier de bioinformatique, Université Pierre et Marie CurieParisFrance
  4. 4.CNRS UMR8186, Département de biologieEcole Normale SupérieureParis cedexFrance
  5. 5.The Sainsbury Laboratory, John Innes CentreNorwichUK

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