Advertisement

Northern Blot Detection of Virus-Derived Small Interfering RNAs in Caenorhabditis elegans Using Nonradioactive Oligo Probes

  • Tianyun Long
  • Rui LuEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1656)

Abstract

Northern blot analysis has been widely used as a tool for detection and characterization of specific RNA molecules. When coupled with radioactive probe northern blot allows for robust detection and characterization of small RNA molecules of trace amount. Here, we describe the detection and size characterization of virus-derived small interfering RNAs (vsiRNAs) in C. elegans using nonradioactive DNA oligo probes in northern blotting. Our protocol allows for the detection and characterization of not only primary vsiRNAs but also secondary vsiRNAs, a class of single-stranded vsiRNAs that has distinct migration pattern, and can be easily adapted to the detection of vsiRNAs in other organisms.

Key words

Northern blot Nonradioactive Virus-derived siRNA DIG-labeled probe PAGE gel electrophoresis Caenorhabditis elegans 

Notes

Acknowledgment

This work was supported by NIH grant R01GM119012-01A1 to R.L.

References

  1. 1.
    Hamilton AJ, Baulcombe DC (1999) A species of small antisense RNA in posttranscriptional gene silencing in plants. Science 286(5441):950–952CrossRefGoogle Scholar
  2. 2.
    Li H, Li WX, Ding SW (2002) Induction and suppression of RNA silencing by an animal virus. Science 296(5571):1319–1321CrossRefGoogle Scholar
  3. 3.
    Lu R et al (2009) An RIG-I-like RNA helicase mediates antiviral RNAi downstream of viral siRNA biogenesis in Caenorhabditis elegans. PLoS Pathog 5(2):e1000286CrossRefGoogle Scholar
  4. 4.
    Ding SW, Voinnet O (2007) Antiviral immunity directed by small RNAs. Cell 130(3):413–426CrossRefGoogle Scholar
  5. 5.
    Roghiyh Aliyari S-WD (2009) RNA-based viral immunity initiated by the Dicer family of host immune receptors. Immunol Rev 227(1):176–188CrossRefGoogle Scholar
  6. 6.
    Guo X, Lu R (2013) Characterization of virus-encoded RNAi suppressors in Caenorhabditis elegans. J Virol 87(10):5414–5423CrossRefGoogle Scholar
  7. 7.
    Guo X et al (2013) Homologous RIG-I–like helicase proteins direct RNAi-mediated antiviral immunity in C. elegans by distinct mechanisms. Proc Natl Acad Sci 110:16085–16090CrossRefGoogle Scholar
  8. 8.
    Guo X et al (2013) Antiviral RNA silencing initiated in the absence of RDE-4, a double-stranded RNA binding protein, in Caenorhabditis elegans. J Virol 87(19):10721–10729CrossRefGoogle Scholar
  9. 9.
    Félix M-A et al (2011) Natural and experimental infection of Caenorhabditis Nematodes by novel viruses related to Nodaviruses. PLoS Biol 9(1):e1000586CrossRefGoogle Scholar
  10. 10.
    Ashe A et al (2013) A deletion polymorphism in the Caenorhabditis elegans RIG-I homolog disables viral RNA dicing and antiviral immunity. elife 2:e00994 CrossRefGoogle Scholar
  11. 11.
    Kennedy S, Wang D, Ruvkun G (2004) A conserved siRNA-degrading RNase negatively regulates RNA interference in C. elegans. Nature 427(6975):645–649CrossRefGoogle Scholar
  12. 12.
    Ghildiyal M, Zamore PD (2009) Small silencing RNAs: an expanding universe. Nat Rev Genet 10(2):94–108CrossRefGoogle Scholar
  13. 13.
    Ramkissoon SH et al (2006) Nonisotopic detection of microRNA using digoxigenin labeled RNA probes. Mol Cell Probes 20(1):1–4CrossRefGoogle Scholar
  14. 14.
    Höltke HJ, Kessler C (1990) Non-radioactive labeling of RNA transcripts in vitro with the hapten digoxigenin (DIG); hybridization and ELISA-based detection. Nucleic Acids Res 18(19):5843–5851CrossRefGoogle Scholar
  15. 15.
    Kim SW et al (2010) A sensitive non-radioactive northern blot method to detect small RNAs. Nucleic Acids Res 38(7):e98–e98CrossRefGoogle Scholar
  16. 16.
    Guo X, Li W-X, Lu R (2012) Silencing of host genes directed by virus-derived short interfering RNAs in Caenorhabditis elegans. J Virol 86(21):11645–11653CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  1. 1.Department of Biological SciencesLouisiana State UniversityBaton RougeUSA

Personalised recommendations