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A universal method for the rapid isolation of all known classes of functional silencing small RNAs

Abstract : Diverse classes of silencing small (s)RNAs operate via ARGONAUTE-family proteins within RNA-induced-silencing-complexes (RISCs). Here, we have streamlined various embodiments of a Q-sepharose-based RISC-purification method that relies on conserved biochemical properties of all ARGONAUTEs. We show, in multiple benchmarking assays, that the resulting 15-min benchtop extraction procedure allows simultaneous purification of all known classes of RISC-associated sRNAs without prior knowledge of the samples-intrinsic ARGONAUTE repertoires. Optimized under a user-friendly format, the method-coined 'TraPR' for Trans-kingdom, rapid, affordable Purification of RISCs-operates irrespectively of the organism, tissue, cell type or bio-fluid of interest , and scales to minute amounts of input material. The method is highly suited for direct profiling of silencing sRNAs, with TraPR-generated sequenc-ing libraries outperforming those obtained via gold-standard procedures that require immunoprecipita-tions and/or lengthy polyacrylamide gel-selection. TraPR considerably improves the quality and consistency of silencing sRNA sample preparation including from notoriously difficult-to-handle tissues/bio-fluids such as starchy storage roots or mammalian plasma, and regardless of RNA contaminants or RNA degradation status of samples.
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Thomas Grentzinger, Stefan Oberlin, Gregory Schott, Dominik Handler, Julia Svozil, et al.. A universal method for the rapid isolation of all known classes of functional silencing small RNAs. Nucleic Acids Research, Oxford University Press, 2020, 48 (14), pp.e79-e79. ⟨10.1093/nar/gkaa472⟩. ⟨hal-02990736⟩

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