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Detection of MicroRNAs by Northern Blot

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MicroRNA Detection and Target Identification

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2630))

Abstract

Small RNAs (sRNAs) are key regulators of transcriptomes and proteomes of organisms through their sequence-specific interaction with complementary RNA targets. sRNAs can be classified according to their origin and mode of action into different classes such as: microRNAs (miRNAs), small interfering RNAs (siRNAs) and PIWI-interacting RNAs (piRNAs). The abundance and specific spatio-temporal expression of many sRNAs, especially miRNAs, is relevant for their biological function. Northern blotting is a widely used technique to study sRNAs because it is quantitative, relatively inexpensive, and readily available for most laboratories. This chapter describes the protocols for radioactive and non-radioactive sRNA Northern blot analysis, which includes RNA extraction, polyacrylamide gel electrophoresis, membrane transfer, hybridisation and detection of sRNA using oligonucleotide probes. The protocol is described to prepare most of the reagents needed in the lab, but also timesaving commercial reagent alternatives are included. Suggestions and nuances obtained from experience are included as Notes.

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References

  1. Yu Y, Zhang Y, Chen X, Chen Y (2019) Plant noncoding RNAs: hidden players in development and stress responses. Annu Rev Cell Dev Biol 35:407–431

    Article  CAS  Google Scholar 

  2. Lee Y, Ahn C, Han J et al (2003) The nuclear RNase III Drosha initiates microRNA processing. Nature 425:415–419

    Article  CAS  Google Scholar 

  3. Tomari Y, Zamore PD (2005) Perspective: machines for RNAi. Genes Dev 19:517–529

    Article  CAS  Google Scholar 

  4. Castel SE, Martienssen RA (2013) RNA interference in the nucleus: roles for small RNAs in transcription, epigenetics and beyond. Nat Rev Genet 14:100–112

    Article  CAS  Google Scholar 

  5. Martinho C, Confraria A, Elias CA et al (2015) Dissection of miRNA pathways using arabidopsis mesophyll protoplasts. Mol Plant 8:261–275

    Article  CAS  Google Scholar 

  6. Ahmed A, Ward NJ, Moxon S et al (2015) A database of microRNA expression patterns in Xenopus laevis. PLoS One 10:e0138313

    Article  Google Scholar 

  7. Lopez-Gomollon S, Beckers M, Rathjen T et al (2014) Global discovery and characterization of small non-coding RNAs in marine microalgae. BMC Genomics 15:697

    Article  Google Scholar 

  8. Mohorianu I, Schwach F, Jing R et al (2011) Profiling of short RNAs during fleshy fruit development reveals stage-specific sRNAome expression patterns. Plant J 67:232–246

    Article  CAS  Google Scholar 

  9. Nicolas FE, Pais H, Schwach F et al (2011) mRNA expression profiling reveals conserved and non-conserved miR-140 targets. RNA Biol 8:607–615

    Article  CAS  Google Scholar 

  10. Nicolas FE, Moxon S, de Haro JP et al (2010) Endogenous short RNAs generated by dicer 2 and RNA-dependent RNA polymerase 1 regulate mRNAs in the basal fungus Mucor circinelloides. Nucleic Acids Res 38:5535–5541

    Article  CAS  Google Scholar 

  11. López-Gomollón S (2011) Detecting sRNAs by northern blotting. Methods Mol Biol 732:25–38

    Article  Google Scholar 

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

    Article  CAS  Google Scholar 

  13. Kumar GNM, Iyer S, Knowles NR (2007) Extraction of RNA from fresh, frozen, and lyophilized tuber and root tissues. J Agric Food Chem 55:1674–1678

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  15. Pall GS, Codony-Servat C, Byrne J et al (2007) Carbodiimide-mediated cross-linking of RNA to nylon membranes improves the detection of siRNA, miRNA and piRNA by northern blot. Nucleic Acids Res 35:e60–e60

    Article  Google Scholar 

  16. Vester B, Wengel J (2004) LNA (locked nucleic acid): high-affinity targeting of complementary RNA and DNA. Biochemistry 43:13233–13241

    Article  CAS  Google Scholar 

  17. Válóczi A, Hornyik C, Varga N et al (2004) Sensitive and specific detection of microRNAs by northern blot analysis using LNA-modified oligonucleotide probes. Nucleic Acids Res 32:e175–e175

    Article  Google Scholar 

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Acknowledgments

SLG is a Senior Broodbank Research Fellow.

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Correspondence to Sara Lopez-Gomollon .

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Martinho, C., Lopez-Gomollon, S. (2023). Detection of MicroRNAs by Northern Blot. In: Dalmay, T. (eds) MicroRNA Detection and Target Identification. Methods in Molecular Biology, vol 2630. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2982-6_4

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  • DOI: https://doi.org/10.1007/978-1-0716-2982-6_4

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2981-9

  • Online ISBN: 978-1-0716-2982-6

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