Northern Blot Detection of Tiny RNAs.

Blotting, Northern Cross-Linking Reagents / chemistry DNA Probes / chemistry Denaturing Gradient Gel Electrophoresis Digoxigenin / chemistry Ethyldimethylaminopropyl Carbodiimide / chemistry Native Polyacrylamide Gel Electrophoresis RNA / analysis
Digoxigenin EDC crosslinking LNA Native PAGE Northern blot UV crosslinking

Journal

Methods in molecular biology (Clifton, N.J.)
ISSN: 1940-6029
Titre abrégé: Methods Mol Biol
Pays: United States
ID NLM: 9214969

Informations de publication

Date de publication:
2021
Historique:
entrez: 1 4 2021
pubmed: 2 4 2021
medline: 23 6 2021
Statut: ppublish

Résumé

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'-

Identifiants

DOI: 10.1007/978-1-0716-1386-3_5 PMID: 33792870
pubmed: 33792870
doi: 10.1007/978-1-0716-1386-3_5
doi:

Substances chimiques

Cross-Linking Reagents 0
DNA Probes 0
RNA 63231-63-0
Digoxigenin NQ1SX9LNAU
Ethyldimethylaminopropyl Carbodiimide RJ5OZG6I4A

Types de publication

Comparative Study Journal Article Research Support, Non-U.S. Gov't

Langues

eng

Sous-ensembles de citation

IM

Pagination

41-58

Références

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doi: 10.1093/nar/gni178
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
doi: 10.1093/nar/gkm112
Várallyay E, Burgyán J, Havelda Z (2008) MicroRNA detection by northern blotting using locked nucleic acid probes. Nat Protoc 3:190–196
doi: 10.1038/nprot.2007.528
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
doi: 10.1093/nar/gkq437
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
doi: 10.1002/9783527647064.ch4
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
doi: 10.1007/978-1-4939-2547-6_4
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
doi: 10.1007/978-1-4939-2547-6_5
Haines AM, Tobe SS, Kobus HJ, Linacre A (2015) Properties of nucleic acid staining dyes used in gel electrophoresis. Electrophoresis 36:941–944
doi: 10.1002/elps.201400496
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
doi: 10.1261/rna.042077.113
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
doi: 10.4161/rna.8.5.16151
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
doi: 10.1016/j.biochi.2014.12.019
Pall GS, Hamilton AJ (2008) Improved northern blot method for enhanced detection of small RNA. Nat Protoc 3:1077–1084
doi: 10.1038/nprot.2008.67
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
doi: 10.1006/abio.1997.2044

Auteurs

Jana C Wiegard (JC)

Institute of Pharmaceutical Chemistry, Philipps-University Marburg, Marburg, Germany.

M Amri C Schlüter (MAC)

Institute of Pharmaceutical Chemistry, Philipps-University Marburg, Marburg, Germany.

Olga Y Burenina (OY)

Center of Life Sciences, Skolkovo Institute of Science and Technology, Moscow, Russia.
Chemistry Department and A.N. Belozersky Institute of Physico-Chemical Biology, M.V. Lomonosov Moscow State University, Moscow, Russia.

Elena A Kubareva (EA)

Chemistry Department and A.N. Belozersky Institute of Physico-Chemical Biology, M.V. Lomonosov Moscow State University, Moscow, Russia.

Gabriele Klug (G)

Institute of Microbiology and Molecular Biology, Justus-Liebig-University-Gießen, Gießen, Germany.

Arnold Grünweller (A)

Institute of Pharmaceutical Chemistry, Philipps-University Marburg, Marburg, Germany.

Roland K Hartmann (RK)

Institute of Pharmaceutical Chemistry, Philipps-University Marburg, Marburg, Germany. roland.hartmann@staff.uni-marburg.de.

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Classifications MeSH

questionsmedicales.fr › Techniques d'investigation › Techniques de sonde moléculaire › Technique de Northern › Northern Blot Detection of Tiny RNAs.
questionsmedicales.fr › Actions chimiques et utilisations › Utilisations spécialisées de produits chimiques › Produits chimiques de laboratoire › Indicateurs et réactifs › Réactifs réticulants › Northern Blot Detection of Tiny RNAs.
questionsmedicales.fr › Actions chimiques et utilisations › Utilisations spécialisées de produits chimiques › Produits chimiques de laboratoire › Sondes moléculaires › Sondes d'acide nucléique › Sondes d'ADN › Northern Blot Detection of Tiny RNAs.
questionsmedicales.fr › Techniques d'investigation › Techniques électrochimiques › Électrophorèse › Électrophorèse sur gel de polyacrylamide › Électrophorèse sur gel en gradient dénaturant › Northern Blot Detection of Tiny RNAs.
questionsmedicales.fr › Composés polycycliques › Composés à cycles fusionnés › Stéroïdes › Cardanolides › Cardénolides › Digoxigénine › Northern Blot Detection of Tiny RNAs.
questionsmedicales.fr › Composés chimiques organiques › Imines › Carbodiimides › 1-Éthyl-3-(3-diméthylaminopropyl)carbodiimide › Northern Blot Detection of Tiny RNAs.
questionsmedicales.fr › Techniques d'investigation › Techniques électrochimiques › Électrophorèse › Électrophorèse sur gel de polyacrylamide › Électrophorèse sur gel de polyacrylamide non dénaturant › Northern Blot Detection of Tiny RNAs.
questionsmedicales.fr › Acides nucléiques, nucléotides et nucléosides › Acides nucléiques › ARN › Northern Blot Detection of Tiny RNAs.