MicroRNA Detection by Whole-Mount In Situ Hybridization in C. elegans

  • Yoshiki AndachiEmail author
  • Yuji Kohara
Part of the Methods in Molecular Biology book series (MIMB, volume 1680)


MicroRNAs (miRNAs) loaded on argonaute proteins guide RNA-induced silencing complexes to target mRNAs. An excellent method to decipher the spatiotemporal expression patterns of miRNAs is whole-mount in situ hybridization (WISH), which has been successfully used in vertebrate embryos but still remains unavailable for many animal species. Here, we describe a WISH method for miRNA detection in Caenorhabditis elegans at both embryonic and post-embryonic stages. Strategies devised for detection include fixation of animals with carbodiimide at a high temperature and subsequent partial digestion of the fixed animals with an extremely high concentration of proteinase. WISH signals are visualized by staining with a chromogenic substrate or a fluorescent dye.

Key words

microRNA In situ hybridization C. elegans Fixation Chromogenic staining Fluorescent staining 



This work was supported by JSPS KAKENHI Grant number 22241047 and 25250025 to Y.K.


  1. 1.
    Wienholds E, Kloosterman WP, Miska E et al (2005) MicroRNA expression in zebrafish embryonic development. Science 309:310–311CrossRefPubMedGoogle Scholar
  2. 2.
    Sokol NS, Ambros V (2005) Mesodermally expressed drosophila microRNA-1 is regulated by twist and is required in muscles during larval growth. Genes Dev 19:2343–2354CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Ason B, Darnell DK, Wittbrodt B et al (2006) Differences in vertebrate microRNA expression. Proc Natl Acad Sci U S A 103:14385–14389CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Darnell DK, Kaur S, Stanislaw S et al (2006) MicroRNA expression during chick embryo development. Dev Dyn 235:3156–3165CrossRefPubMedGoogle Scholar
  5. 5.
    Kloosterman WP, Wienholds E, de Bruijn E et al (2006) In situ detection of miRNAs in animal embryos using LNA-modified oligonucleotide probes. Nat Methods 3:27–29CrossRefPubMedGoogle Scholar
  6. 6.
    Petersen M, Wengel J (2003) LNA: a versatile tool for therapeuticsand genomics. Trends Biotechnol 21:74–81CrossRefPubMedGoogle Scholar
  7. 7.
    Pena JT, Sohn-Lee C, Rouhanifard SH et al (2009) miRNA in situ hybridization in formaldehyde and EDC-fixed tissues. Nat Methods 6:139–141CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Renwick N, Cekan P, Masry PA et al (2013) Multicolor microRNA FISH effectively differentiates tumor types. J Clin Invest 123:2694–2702CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Sung HW, Chang WH, Ma CY et al (2003) Crosslinking of biological tissues using genipin and/or carbodiimide. J Biomed Mater Res 64A:427–438CrossRefGoogle Scholar
  10. 10.
    Andachi Y, Kohara Y (2016) A whole-mount in situ hybridization method for microRNA detection in Caenorhabditis elegans. RNA 22:1099–1106CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Tabara H, Motohashi T, Kohara Y (1996) A multi-well version of in situ hybridization on whole mount embryos of Caenorhabditis elegans. Nucleic Acids Res 24:2119–2124CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Motohashi T, Tabara H, Kohara Y (2006) Protocols for large scale in situ hybridization on C. elegans larvae. In: The C. elegans research community (ed) WormBook. Accessed 24 Jul 2006
  13. 13.
    Motohashi T, Hirono K, Kohara Y (2015) In situ hybridization on whole mount embryos of C. elegans. In: The C. elegans research community (ed) WormBook. Accessed 19 May 2015
  14. 14.
    McEwen TJ, Yao Q, Yun S et al (2016) Small RNA in situ hybridization in Caenorhabditis elegans, combined with RNA-seq, identifies germline-enriched microRNAs. Dev Biol 418:248–257CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2018

Authors and Affiliations

  1. 1.Genetic Strains Research CenterNational Institute of Genetics, Research Organization of Information and SystemsMishimaJapan
  2. 2.Department of GeneticsSOKENDAI (The Graduate University for Advanced Studies)MishimaJapan

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