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Improved Denaturation of Small RNA Duplexes and Its Application for Northern Blotting

  • C. Jake Harris
  • David C. Baulcombe
  • Attila MolnarEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1580)

Abstract

Small RNAs (sRNAs) are short (18–30 nucleotide) noncoding RNA molecules, which control gene expression and pathogen response in eukaryotes. They are associated with and guide nucleases to target nucleic acids by nucleotide base pairing. We found that current techniques for small RNA detection are adversely affected by the presence of complementary RNA. Thus we established FDF-PAGE (fully denaturing formaldehyde polyacrylamide gel electrophoresis), which dramatically improves denaturation efficiency and subsequently the detection of sequestered sRNAs.

Key words

Small RNA Sequestration Polyacrylamide gel electrophoresis Denaturation Improved detection 

Notes

Acknowledgment

C.J.H was supported by a BBSRC PhD Studentship. D.C.B. is the Royal Society Edward Penley Abraham Research Professor. This work was supported by the ERC Advanced Investigator grant ERC-2013-AdG 340642 TRIBE. A.M. is a Chancellor’s Fellow at the University of Edinburgh.

References

  1. 1.
    Hamilton AJ, Baulcombe DC (1999) A species of small antisense RNA in posttranscriptional gene silencing in plants. Science 286:950–952CrossRefPubMedGoogle Scholar
  2. 2.
    Zamore PD, Tuschl T, Sharp PA, Bartel DP (2000) RNAi: double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals. Cell 101:25–33CrossRefPubMedGoogle Scholar
  3. 3.
    Molnár A, Schwach F, Studholme DJ, Thuenemann EC, Baulcombe DC (2007) miRNAs control gene expression in the single-cell alga Chlamydomonas reinhardtii. Nature 447:1126–1129CrossRefPubMedGoogle Scholar
  4. 4.
    Terry B, Karol M, Du T (2004) Analysis of RNA by Northern and slot blot. Curr Protoc Mol Biol Chapter 4Google Scholar
  5. 5.
    Smith NA, Eamens AL, Wang M-B (2010) The presence of high-molecular-weight viral RNAs interferes with the detection of viral small RNAs. RNA 16:1062–1067CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Harris CJ, Molnar A, Muller SY, Baulcombe DC (2015) FDF-PAGE: a powerful technique revealing previously undetected small RNAs sequestered by complementary transcripts. Nucleic Acids Res 43:7590–7599CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Green MR, Sambrook J (2012) Molecular cloning: a laboratory manual, 4th edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor. ISBN: 978-1-936113-42-2Google Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • C. Jake Harris
    • 1
  • David C. Baulcombe
    • 1
  • Attila Molnar
    • 2
    Email author
  1. 1.Department of Plant SciencesUniversity of CambridgeCambridgeUK
  2. 2.School of Biological SciencesUniversity of EdinburghEdinburghUK

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