Skip to main content
SpringerLink
Log in
Book cover

Plant Epigenetics pp 141–162Cite as

Northern Blotting Techniques for Small RNAs

  • Protocol
  • First Online:

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

Abstract

Cells have evolved intricate RNA-directed mechanisms that destroy viruses, silence transposons, and regulate gene expression. These nucleic acid surveillance and gene silencing mechanisms rely upon the selective base-pairing of ~19–25 nt small RNAs to complementary RNA targets. This chapter describes northern blot hybridization techniques for the detection of such small RNAs. Blots spiked with synthetic standards are used to illustrate the detection specificity and sensitivity of DNA oligonucleotide probes. Known endogenous small RNAs are then analyzed in samples prepared from several model plants, including Arabidopsis thaliana, Nicotiana benthamiana, Oryza sativa, Zea mays, and Physcomitrella patens, as well as from the animals Drosophila melanogaster and Mus musculus. Finally, the value of northern blotting for dissecting small RNA biogenesis is shown using an example of virus infection in A. thaliana.

This is a preview of subscription content, log in via an institution.

Protocol
USD   49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD   89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD   119.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD   159.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Learn about institutional subscriptions

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

References

  1. Sambrook J, Russell DW (2001) Molecular cloning. A laboratory manual, 3rd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor

    Google Scholar 

  2. Southern EM (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 98(3):503–517

    Article  CAS  PubMed  Google Scholar 

  3. Southern E (2006) Southern blotting. Nat Protoc 1(2):518–525

    Article  CAS  PubMed  Google Scholar 

  4. 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 U S A 74(12):5350–5354

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Thomas PS (1980) Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc Natl Acad Sci U S A 77(9):5201–5205

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Brown T, Mackey K, Du T (2004) Analysis of RNA by northern and slot blot hybridization. Curr Protoc Mol Biol 4:49

    Google Scholar 

  7. Hamilton AJ, Baulcombe DC (1999) A species of small antisense RNA in posttranscriptional gene silencing in plants. Science 286(5441):950–952

    Article  CAS  PubMed  Google Scholar 

  8. Hutvagner G, Mlynarova L, Nap JP (2000) Detailed characterization of the posttranscriptional gene-silencing-related small RNA in a GUS gene-silenced tobacco. RNA 6(10):1445–1454

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Reinhart BJ, Weinstein EG, Rhoades MW, Bartel B, Bartel DP (2002) MicroRNAs in plants. Genes Dev 16(13):1616–1626

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Lau NC, Lim LP, Weinstein EG, Bartel DP (2001) An abundant class of tiny RNAs with probable regulatory roles in Caenorhabditis elegans. Science 294(5543):858–862

    Article  CAS  PubMed  Google Scholar 

  11. Llave C, Kasschau KD, Rector MA, Carrington JC (2002) Endogenous and silencing-associated small RNAs in plants. Plant Cell 14(7):1605–1619

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Lagos-Quintana M, Rauhut R, Lendeckel W, Tuschl T (2001) Identification of novel genes coding for small expressed RNAs. Science 294(5543):853–858

    Article  CAS  PubMed  Google Scholar 

  13. Pfeffer S, Zavolan M, Grasser FA, Chien M, Russo JJ, Ju J, John B, Enright AJ, Marks D, Sander C, Tuschl T (2004) Identification of virus-encoded microRNAs. Science 304(5671):734–736

    Article  CAS  PubMed  Google Scholar 

  14. Gu W, Shirayama M, Conte D Jr, Vasale J, Batista PJ et al (2009) Distinct argonaute-mediated 22G-RNA pathways direct genome surveillance in the C. elegans germline. Mol Cell 36(2):231–244

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Chalker DL, Fuller P, Yao MC (2005) Communication between parental and developing genomes during tetrahymena nuclear differentiation is likely mediated by homologous RNAs. Genetics 169(1):149–160

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Girard A, Sachidanandam R, Hannon GJ, Carmell MA (2006) A germline-specific class of small RNAs binds mammalian Piwi proteins. Nature 442(7099):199–202

    PubMed  Google Scholar 

  17. Pane A, Wehr K, Schupbach T (2007) zucchini and squash encode two putative nucleases required for rasiRNA production in the Drosophila germline. Dev Cell 12(6):851–862

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Meins F Jr, Si-Ammour A, Blevins T (2005) RNA silencing systems and their relevance to plant development. Annu Rev Cell Dev Biol 21:297–318

    Article  CAS  PubMed  Google Scholar 

  19. Chapman EJ, Carrington JC (2007) Specialization and evolution of endogenous small RNA pathways. Nat Rev Genet 8(11):884–896

    Article  CAS  PubMed  Google Scholar 

  20. Pikaard CS, Haag JR, Pontes OM, Blevins T, Cocklin R (2012) A transcription fork model for Pol IV and Pol V-dependent RNA-directed DNA methylation. Cold Spring Harb Symp Quant Biol 77:205–212

    Article  CAS  PubMed  Google Scholar 

  21. Xie Z, Johansen LK, Gustafson AM, Kasschau KD, Lellis AD, Zilberman D, Jacobsen SE, Carrington JC (2004) Genetic and functional diversification of small RNA pathways in plants. PLoS Biol 2(5), E104

    Article  PubMed  PubMed Central  Google Scholar 

  22. Gasciolli V, Mallory AC, Bartel DP, Vaucheret H (2005) Partially redundant functions of Arabidopsis DICER-like enzymes and a role for DCL4 in producing trans-acting siRNAs. Curr Biol 15(16):1494–1500

    Article  CAS  PubMed  Google Scholar 

  23. Dunoyer P, Himber C, Voinnet O (2005) DICER-LIKE 4 is required for RNA interference and produces the 21-nucleotide small interfering RNA component of the plant cell-to-cell silencing signal. Nat Genet 37(12):1356–1360

    Article  CAS  PubMed  Google Scholar 

  24. Zilberman D, Cao X, Jacobsen SE (2003) ARGONAUTE4 control of locus-specific siRNA accumulation and DNA and histone methylation. Science 299(5607):716–719

    Article  CAS  PubMed  Google Scholar 

  25. Mette MF, Aufsatz W, van der Winden J, Matzke MA, Matzke AJ (2000) Transcriptional silencing and promoter methylation triggered by double-stranded RNA. EMBO J 19(19):5194–5201

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Vazquez F, Vaucheret H, Rajagopalan R, Lepers C, Gasciolli V, Mallory AC, Hilbert JL, Bartel DP, Crete P (2004) Endogenous trans-acting siRNAs regulate the accumulation of Arabidopsis mRNAs. Mol Cell 16(1):69–79

    Article  CAS  PubMed  Google Scholar 

  27. Yu B, Yang Z, Li J, Minakhina S, Yang M, Padgett RW, Steward R, Chen X (2005) Methylation as a crucial step in plant microRNA biogenesis. Science 307(5711):932–935

    Article  CAS  PubMed  Google Scholar 

  28. Vazquez F, Gasciolli V, Crete P, Vaucheret H (2004) The nuclear dsRNA binding protein HYL1 is required for microRNA accumulation and plant development, but not posttranscriptional transgene silencing. Curr Biol 14(4):346–351

    Article  CAS  PubMed  Google Scholar 

  29. Onodera Y, Haag JR, Ream T, Nunes PC, Pontes O, Pikaard CS (2005) Plant nuclear RNA polymerase IV mediates siRNA and DNA methylation-dependent heterochromatin formation. Cell 120(5):613–622

    Article  CAS  PubMed  Google Scholar 

  30. Hutvagner G, McLachlan J, Pasquinelli AE, Balint E, Tuschl T, Zamore PD (2001) A cellular function for the RNA-interference enzyme Dicer in the maturation of the let-7 small temporal RNA. Science 293(5531):834–838

    Article  CAS  PubMed  Google Scholar 

  31. Reinhart BJ, Slack FJ, Basson M, Pasquinelli AE, Bettinger JC, Rougvie AE, Horvitz HR, Ruvkun G (2000) The 21-nucleotide let-7 RNA regulates developmental timing in Caenorhabditis elegans. Nature 403(6772):901–906

    Article  CAS  PubMed  Google Scholar 

  32. Pasquinelli AE, Reinhart BJ, Slack F, Martindale MQ, Kuroda MI et al (2000) Conservation of the sequence and temporal expression of let-7 heterochronic regulatory RNA. Nature 408(6808):86–89

    Article  CAS  PubMed  Google Scholar 

  33. Grishok A, Pasquinelli AE, Conte D, Li N, Parrish S, Ha I et al (2001) Genes and mechanisms related to RNA interference regulate expression of the small temporal RNAs that control C. elegans developmental timing. Cell 106(1):23–34

    Article  CAS  PubMed  Google Scholar 

  34. Ketting RF, Fischer SE, Bernstein E, Sijen T, Hannon GJ, Plasterk RH (2001) Dicer functions in RNA interference and in synthesis of small RNA involved in developmental timing in C. elegans. Genes Dev 15(20):2654–2659

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Bartel DP (2004) MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116(2):281–297

    Article  CAS  PubMed  Google Scholar 

  36. Axtell MJ (2008) Evolution of microRNAs and their targets: Are all microRNAs biologically relevant? Biochim Biophys Acta 1779(11):725–734

    Article  CAS  PubMed  Google Scholar 

  37. Jones-Rhoades MW, Bartel DP, Bartel B (2006) MicroRNAS and their regulatory roles in plants. Annu Rev Plant Biol 57:19–53

    Article  CAS  PubMed  Google Scholar 

  38. Kutter C, Schob H, Stadler M, Meins F Jr, Si-Ammour A (2007) MicroRNA-mediated regulation of stomatal development in Arabidopsis. Plant Cell 19(8):2417–2429

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Rajagopalan R, Vaucheret H, Trejo J, Bartel DP (2006) A diverse and evolutionarily fluid set of microRNAs in Arabidopsis thaliana. Genes Dev 20(24):3407–3425

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Bologna NG, Voinnet O (2014) The diversity, biogenesis, and activities of endogenous silencing small RNAs in Arabidopsis. Annu Rev Plant Biol 65:473–503

    Article  CAS  PubMed  Google Scholar 

  41. Holoch D, Moazed D (2015) RNA-mediated epigenetic regulation of gene expression. Nat Rev Genet 16(2):71–84

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Pikaard CS, Mittelsten Scheid O (2014) Epigenetic regulation in plants. Cold Spring Harb Perspect Biol 6(12):a019315

    Article  PubMed  Google Scholar 

  43. Blevins T, Pontvianne F, Cocklin R, Podicheti R, Chandrasekhara C, Yerneni S et al (2014) A two-step process for epigenetic inheritance in Arabidopsis. Mol Cell 54(1):30–42

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Henderson IR, Jacobsen SE (2007) Epigenetic inheritance in plants. Nature 447(7143):418–424

    Article  CAS  PubMed  Google Scholar 

  45. Head SR, Komori HK, LaMere SA, Whisenant T, Van Nieuwerburgh F, Salomon DR, Ordoukhanian P (2014) Library construction for next-generation sequencing: overviews and challenges. Biotechniques 56(2):61–64, 66, 68, passim

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Coruh C, Shahid S, Axtell MJ (2014) Seeing the forest for the trees: annotating small RNA producing genes in plants. Curr Opin Plant Biol 18:87–95

    Article  PubMed  Google Scholar 

  47. Carbonell A, Fahlgren N, Garcia-Ruiz H, Gilbert KB, Montgomery TA, Nguyen T, Cuperus JT, Carrington JC (2012) Functional analysis of three Arabidopsis ARGONAUTES using slicer-defective mutants. Plant Cell 24(9):3613–3629

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Blevins T, Rajeswaran R, Aregger M, Borah BK, Schepetilnikov M, Baerlocher L et al (2011) Massive production of small RNAs from a non-coding region of Cauliflower mosaic virus in plant defense and viral counter-defense. Nucleic Acids Res 39(12):5003–5014

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Mari-Ordonez A, Marchais A, Etcheverry M, Martin A, Colot V, Voinnet O (2013) Reconstructing de novo silencing of an active plant retrotransposon. Nat Genet 45(9):1029–1039

    Article  CAS  PubMed  Google Scholar 

  50. Reimao-Pinto MM, Ignatova V, Burkard TR, Hung JH, Manzenreither RA, Sowemimo I, Herzog VA, Reichholf B, Farina-Lopez S, Ameres SL (2015) Uridylation of RNA hairpins by tailor confines the emergence of MicroRNAs in drosophila. Mol Cell 59(2):203–216

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Reed KC, Mann DA (1985) Rapid transfer of DNA from agarose gels to nylon membranes. Nucleic Acids Res 13(20):7207–7221

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Alonso JM, Stepanova AN, Leisse TJ, Kim CJ, Chen H, Shinn P et al (2003) Genome-wide insertional mutagenesis of Arabidopsis thaliana. Science 301(5633):653–657

    Article  PubMed  Google Scholar 

  53. Sessions A, Burke E, Presting G, Aux G, McElver J, Patton D et al (2002) A high-throughput Arabidopsis reverse genetics system. Plant Cell 14(12):2985–2994

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Blevins T, Rajeswaran R, Shivaprasad PV, Beknazariants D, Si-Ammour A, Park HS et al (2006) Four plant dicers mediate viral small RNA biogenesis and DNA virus induced silencing. Nucleic Acids Res 34(21):6233–6246

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Chomczynski P, Sacchi N (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162(1):156–159

    Article  CAS  PubMed  Google Scholar 

  56. Chomczynski P, Sacchi N (2006) The single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction: twenty-something years on. Nat Protoc 1(2):581–585

    Article  CAS  PubMed  Google Scholar 

  57. Pall GS, Hamilton AJ (2008) Improved northern blot method for enhanced detection of small RNA. Nat Protoc 3(6):1077–1084

    Article  CAS  PubMed  Google Scholar 

  58. Pontes O, Li CF, Nunes PC, Haag J, Ream T, Vitins A, Jacobsen SE, Pikaard CS (2006) The Arabidopsis chromatin-modifying nuclear siRNA pathway involves a nucleolar RNA processing center. Cell 126(1):79–92

    Article  CAS  PubMed  Google Scholar 

  59. Varallyay E, Burgyan J, Havelda Z (2008) MicroRNA detection by northern blotting using locked nucleic acid probes. Nat Protoc 3(2):190–196

    Article  CAS  PubMed  Google Scholar 

  60. Henderson IR, Jacobsen SE (2008) Tandem repeats upstream of the Arabidopsis endogene SDC recruit non-CG DNA methylation and initiate siRNA spreading. Genes Dev 22(12):1597–1606

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Zheng B, Wang Z, Li S, Yu B, Liu JY, Chen X (2009) Intergenic transcription by RNA polymerase II coordinates Pol IV and Pol V in siRNA-directed transcriptional gene silencing in Arabidopsis. Genes Dev 23(24):2850–2860

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. Castoldi M, Schmidt S, Benes V, Noerholm M, Kulozik AE, Hentze MW, Muckenthaler MU (2006) A sensitive array for microRNA expression profiling (miChip) based on locked nucleic acids (LNA). RNA 12(5):913–920

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  63. Bonifacio GF, Brown T, Conn GL, Lane AN (1997) Comparison of the electrophoretic and hydrodynamic properties of DNA and RNA oligonucleotide duplexes. Biophys J 73(3):1532–1538

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. Aravin AA, Lagos-Quintana M, Yalcin A, Zavolan M, Marks D, Snyder B, Gaasterland T, Meyer J, Tuschl T (2003) The small RNA profile during Drosophila melanogaster development. Dev Cell 5(2):337–350

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

Many thanks to Frederick Meins, Jr. and Craig Pikaard for providing support and facilities for experiments shown in this chapter. Azeddine Si-Ammour and Hanspeter Schöb refined the northern blot techniques described here. Mikhail Pooggin and Thomas Hohn provided materials for the viral experiments. Mike Dyer cared for leafy plants , Pierre-François Perroud provided moss tissue, and Kathryn Huisinga supplied Drosophila embryos. Tatiana Simon and Luciano Marpegan provided mouse liver. Franck Vazquez, Mikhail Pooggin, and Andrzej Wierzbicki provided critical comments on the first edition of this book chapter. This work was supported by a Friedrich Miescher Institute student fellowship, and postdoctoral fellowships from the Swiss National Foundation and Novartis Foundation.

Author information

Authors and Affiliations

  1. Institut de Biologie Moléculaire des Plantes, Centre National de la Recherche Scientifique (CNRS) UPR2357, 12 rue du Général Zimmer, Strasbourg Cedex, 67084, USA

    Todd Blevins

Authors
  1. Todd Blevins
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Todd Blevins .

Editor information

Editors and Affiliations

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

    Igor Kovalchuk

Rights and permissions

Reprints and permissions

Copyright information

© 2017 Springer Science+Business Media New York

About this protocol

Cite this protocol

Blevins, T. (2017). Northern Blotting Techniques for Small RNAs. 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_12

Download citation

  • DOI: https://doi.org/10.1007/978-1-4899-7708-3_12

  • Published:

  • Publisher Name: Humana Press, Boston, MA

  • Print ISBN: 978-1-4899-7706-9

  • Online ISBN: 978-1-4899-7708-3

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation