Skip to main content

Northern Blot Detection of Tiny RNAs

  • Protocol
  • First Online:
Small Non-Coding RNAs


Successful detection of very small RNAs (tiny RNAs, ~8–15 nt in length) by northern blotting depends on tailored protocols with respect to transfer and immobilization on membranes as well as design of sensitive detection probes. For RNA crosslinking to positively charged membranes, we compared UV light with chemical RNA crosslinking by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), using either denaturing or native polyacrylamide gels. We show that northern blot detection of tiny RNAs with 5′-digoxigenin-labeled DNA/LNA mixmer probes is a highly sensitive and specific method and, in our hands, more sensitive than using a corresponding DNA/LNA mixmer probe with a 5′-32P-end-label. Furthermore, we provide a robust protocol for northern blot analysis of noncoding RNAs of intermediate size (~50–400 nt).

Jana C. Wiegard and M. Amri C. Schlüter are joint first authors.

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

Access this chapter

Subscribe and save

Springer+ Basic
EUR 32.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or Ebook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
USD 99.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 129.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 199.99
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

Institutional subscriptions

Similar content being viewed by others


  1. Chen C, Ridzon DA, Broomer AJ, Zhou Z, Lee DH, Nguyen JT, Barbisin M, Xu NL, Mahuvakar VR, Andersen MR, Lao KQ, Livak KJ, Guegler KJ (2005) Real-time quantification of microRNAs by stem-loop RT-PCR. Nucleic Acids Res 33:e179

    Article  Google Scholar 

  2. Pall GS, Codony-Servat C, Byrne J, Ritchie L, Hamilton A (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

    Article  Google Scholar 

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

    Article  Google Scholar 

  4. Beckmann BM, Grünweller A, Weber MH, Hartmann RK (2010) Northern blot detection of endogenous small RNAs (approximately14 nt) in bacterial total RNA extracts. Nucleic Acids Res 38:e147

    Article  Google Scholar 

  5. Beckmann BM, Grünweller A, Hartmann RK (2014) Northern blot detection of endogenous small RNAs. In: Hartmann RK, Bindereif A, Schön A, Westhof E (eds) Handbook of RNA biochemistry, 2nd edn. Wiley-VCH, Weinheim, pp 89–103

    Chapter  Google Scholar 

  6. Damm K, Bach S, Müller KM, Klug G, Burenina OY, Kubareva EA, Grünweller A, Hartmann RK (2015) Impact of RNA isolation protocols on RNA detection by northern blotting. Methods Mol Biol (Clifton, NJ) 1296:29–38

    Article  CAS  Google Scholar 

  7. Damm K, Bach S, Müller KM, Klug G, Burenina OY, Kubareva EA, Grünweller A, Hartmann RK (2015) Improved northern blot detection of small RNAs using EDC crosslinking and DNA/LNA probes. Methods Mol Biol (Clifton, NJ) 1296:41–51

    Article  CAS  Google Scholar 

  8. Haines AM, Tobe SS, Kobus HJ, Linacre A (2015) Properties of nucleic acid staining dyes used in gel electrophoresis. Electrophoresis 36:941–944

    Article  CAS  Google Scholar 

  9. Burenina OY, Hoch PG, Damm K, Salas M, Zatsepin TS, Lechner M, Oretskaya TS, Kubareva EA, Hartmann RK (2014) Mechanistic comparison of Bacillus subtilis 6S-1 and 6S-2 RNAs - commonalities and differences. RNA 20:348–359

    Article  CAS  Google Scholar 

  10. Beckmann BM, Burenina OY, Hoch PG, Kubareva EA, Sharma CM, Hartmann RK (2011) In vivo and in vitro analysis of 6S RNA-templated short transcripts in Bacillus subtilis. RNA Biol 8:839–849

    Article  CAS  Google Scholar 

  11. Hoch PG, Burenina OY, Weber MH, Elkina DA, Nesterchuk MV, Sergiev PV, Hartmann RK, Kubareva EA (2015) Phenotypic characterization and complementation analysis of Bacillus subtilis 6S RNA single and double deletion mutants. Biochimie 117:87–99

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  13. Grünweller A, Müller PK (1997) Low background and short exposure times in mapping of DNaseI-hypersensitive sites using digoxigenin-labeled probes. Anal Biochem 247:172–175

    Article  Google Scholar 

Download references


This work was supported by the Deutsche Forschungsgemeinschaft (RTG 2355) to RKH and the Russian Foundation for Basic Research (19-04-00791) to OYB and EAK.

Author information

Authors and Affiliations


Corresponding author

Correspondence to Roland K. Hartmann .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2021 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Wiegard, J.C. et al. (2021). Northern Blot Detection of Tiny RNAs. In: Rederstorff, M. (eds) Small Non-Coding RNAs. Methods in Molecular Biology, vol 2300. Humana, New York, NY.

Download citation

  • DOI:

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1385-6

  • Online ISBN: 978-1-0716-1386-3

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics