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.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Hamilton AJ, Baulcombe DC (1999) A species of small antisense RNA in posttranscriptional gene silencing in plants. Science 286:950–952
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 101:25–33
Molnár A, Schwach F, Studholme DJ, Thuenemann EC, Baulcombe DC (2007) miRNAs control gene expression in the single-cell alga Chlamydomonas reinhardtii. Nature 447:1126–1129
Terry B, Karol M, Du T (2004) Analysis of RNA by Northern and slot blot. Curr Protoc Mol Biol Chapter 4
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 16:1062–1067
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 43:7590–7599
Green MR, Sambrook J (2012) Molecular cloning: a laboratory manual, 4th edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor. ISBN: 978-1-936113-42-2
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.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2017 Springer Science+Business Media LLC
About this protocol
Cite this protocol
Harris, C.J., Baulcombe, D.C., Molnar, A. (2017). Improved Denaturation of Small RNA Duplexes and Its Application for Northern Blotting. In: Dalmay, T. (eds) MicroRNA Detection and Target Identification. Methods in Molecular Biology, vol 1580. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6866-4_1
Download citation
DOI: https://doi.org/10.1007/978-1-4939-6866-4_1
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-6864-0
Online ISBN: 978-1-4939-6866-4
eBook Packages: Springer Protocols