A user-friendly chromatographic method to purify small regulatory RNAs

The discovery of the small regulatory RNA populations has changed our vision of cellular regulations. Indeed, loaded on Argonaute proteins they formed ribonucleoprotein complexes that target complementary sequences and achieved widespread silencing mechanisms conserved in most eukaryotes. The recent development of deep sequencing approaches highly contributed to their detection. Small RNA isolation form 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 purposed as an alternative to the standard size-isolation purification procedure. However, the eventual biases of such a method have been poorly investigated. Moreover, this strategy not only relies on advanced technical skills and expensive material but is time consuming and requires an elevated starting biological material amount. Using bioinformatic comparative analysis of six independent small RNA-sequencing libraries of Drosophila ovaries, we here demonstrate that anion-exchange chromatography purification prior to small RNA extraction unbiasedly enriches datasets in bona fide reads (small regulatory RNA reads) and depletes endogenous contaminants (ribosomal RNAs and degradation products). The resulting increase of sequencing depth provides a major benefit to study rare populations. We then developed a fast and basic manual procedure to purify loaded 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. Overall design: Comparison of small regulatory RNA populations obtained after three different small RNA purification procedures

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Grentzinger T, Armenise C, Pelisson A, Brun C, Mugat B, Chambeyron S