Collection Citation: Jonathan Houseley, Cristina Cruz 2021. Protocols for Northern Analysis of Exosome Substrates and Other Noncoding RNAs. protocols.io https://dx.doi.org/10.17504/protocols.io.bntcmeiw
Manuscript citation:
Cruz C., Houseley J. (2020) Protocols for Northern Analysis of Exosome Substrates and Other Noncoding RNAs. In: LaCava J., Vaňáčová Š. (eds) The Eukaryotic RNA Exosome. Methods in Molecular Biology, vol 2062. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9822-7_5
License: This is an open access collection distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: October 22, 2020
Last Modified: September 02, 2021
Collection Integer ID: 43588
Keywords: lncRNA, ncRNA, northern blot, hybridization, probes, noncoding rna, rna on agarose gel, other noncoding rnas over the past decade, glyoxylated rna, other noncoding rna, processing of ncrna, rna on polyacrylamide, unsurpassed insights into ncrna distribution, rna research, rna, detailed insights into complex rna population, explosion in rna research, complex rna population, specialist rna research group, ncrna distribution, exosome substrate, ncrna, exosome, using standard laboratory electrophoresis equipment, standard laboratory electrophoresis equipment, molecular biology lab, urea gel, agarose gel,
Abstract
Over the past decade a plethora of noncoding RNAs (ncRNAs) have been identified, initiating an explosion in RNA research. Although RNA sequencing methods provide unsurpassed insights into ncRNA distribution and expression, detailed information on structure and processing are harder to extract from sequence data. In contrast, northern blotting methods provide uniquely detailed insights into complex RNA populations but are rarely employed outside specialist RNA research groups. Such techniques are generally considered difficult for nonspecialists, which is unfortunate as substantial technical advances in the past few decades have solved the major challenges. Here we present simple, reproducible and highly robust protocols for separating glyoxylated RNA on agarose gels and heat denatured RNA on polyacrylamide–urea gels using standard laboratory electrophoresis equipment. We also provide reliable transfer and hybridization protocols that do not require optimization for most applications. Together, these should allow any molecular biology lab to elucidate the structure and processing of ncRNAs of interest.
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