title: A user-friendly chromatographic method to purify small regulatory RNAs.
abstract: The discovery of the small regulatory RNAs has changed our vision of cellular regulations. Indeed, when loaded on Argonaute proteins they form ribonucleoprotein complexes (RNPs) that target complementary sequences to achieve widespread silencing mechanisms conserved in most eukaryotes. The recent development of deep sequencing approaches highly contributed to their detection. Small RNA isolation from cells and/or tissues remains a crucial stage to generate robust and relevant sequencing data. In 2006, a novel strategy based on anion-exchange chromatography has been proposed as an alternative to the standard size-isolation purification procedure. Using bioinformatic comparative analysis, we here demonstrate that anion-exchange chromatographic RNP purification prior to small RNA extraction unbiasedly enriches datasets in bona fide reads (small regulatory RNA sequences) and depletes endogenous contaminants (ribosomal RNAs and degradation RNA products). The resulting increase in sequencing depth provides a major benefit to study rare populations. We then developed a fast and basic manual procedure to purify such small non-coding RNAs using anion-exchange chromatography at the bench. We validated the efficiency of this new method and used this strategy to purify small RNAs from various tissues and organisms. We moreover determined that our manual purification increases the output of the previously described anion-exchange chromatography procedure.
authors.affiliation: Institut de Génétique Humaine, CNRS, UPR1142, 34396 Montpellier Cedex 5, France.
authors.forename: Thomas
authors.initials: T
authors.lastname: Grentzinger
authors.affiliation: Institut de Génétique Humaine, CNRS, UPR1142, 34396 Montpellier Cedex 5, France.
authors.forename: Claudia
authors.initials: C
authors.lastname: Armenise
authors.affiliation: Institut de Génétique Humaine, CNRS, UPR1142, 34396 Montpellier Cedex 5, France.
authors.forename: Alain
authors.initials: A
authors.lastname: Pelisson
authors.affiliation: Institut de Génétique Humaine, CNRS, UPR1142, 34396 Montpellier Cedex 5, France.
authors.forename: Christine
authors.initials: C
authors.lastname: Brun
authors.affiliation: Institut de Génétique Humaine, CNRS, UPR1142, 34396 Montpellier Cedex 5, France.
authors.forename: Bruno
authors.initials: B
authors.lastname: Mugat
authors.affiliation: Institut de Génétique Humaine, CNRS, UPR1142, 34396 Montpellier Cedex 5, France. Electronic address: severine.chambeyron@igh.cnrs.fr.
authors.forename: Séverine
authors.initials: S
authors.lastname: Chambeyron
mesh_terms: D000818:Animals; D002852:Chromatography, Ion Exchange; D004330:Drosophila / Q000235:genetics; D005260:Female; D017344:Genes, Insect; D057809:HEK293 Cells; D006801:Humans; D008297:Male; D008810:Mice, Inbred C57BL; D010053:Ovary / Q000378:metabolism; D058727:RNA, Small Untranslated / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D017423:Sequence Analysis, RNA; D013737:Testis / Q000378:metabolism
chemical_list: D058727:RNA, Small Untranslated
keywords: Anion-exchange chromatography; Bioinformatics; Drosophila; High-throughput RNA sequencing; RNA silencing; Small non-coding RNAs