A universal, benchtop, gel-free method for the rapid and simultaneous isolation of all known classes of functional small RNAs

All known silencing small (s)RNAs operate via ARGONAUTE(AGO)-family proteins within RNA-induced-silencing-complexes (RISCs). Based on AGOs conserved biochemical properties, we have developed a universal, 15-min benchtop extraction procedure allowing simultaneous purification of all classes of RISC-associated sRNAs known to date, without prior knowledge of the samples-intrinsic AGO repertoires. Optimized into a user-friendly kit, 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, including from minute amounts of input material. The method is highly suited for direct sRNA deep-sequencing, with TrAPR-generated libraries being qualitatively and quantitatively at least on-par with those obtained via gold-standard procedures involving tedious polyacrylamide gel excisions. TraPR considerably improves the quality and consistency of sRNA sample preparation including from notoriously difficult-to-handle tissues/bio-fluids such as starchy storage roots and mammalian plasma, and regardless of RNA contaminants or samples’ RNA-degradation status. Trans-kingdom, rapid, affordable Purification of RISC (TraPR) protocol for sRNA isolation was compared to standard sRNA library preparation from total RNA. A variety of source tissue, like Arabidopsis flower buds, Drosophila ovaries and mouse livers and plasa were used for these comaparisons.

目前已报道的所有沉默小RNA（silencing small RNAs，sRNAs）均通过RNA诱导沉默复合体（RNA-induced-silencing-complexes，RISCs）内的ARGONAUTE（AGO）家族蛋白发挥功能。基于AGO蛋白保守的生化特性，我们开发了一套通用的15分钟台式提取流程，可在无需预先知晓样本内在AGO蛋白组的前提下，同步纯化当前已知的所有类型与RISC结合的sRNA。该方法已优化为易用型试剂盒，被命名为跨物种快速低成本RISC纯化法（Trans-kingdom, rapid, affordable Purification of RISCs，简称TraPR），可不受研究对象的物种、组织、细胞类型或生物流体限制，且适用于极微量起始材料。该方法非常适配直接开展sRNA深度测序，通过TraPR构建的文库在质量与定量性能上至少可与依赖繁琐聚丙烯酰胺凝胶切胶的金标准流程所获文库相媲美。TraPR可显著提升sRNA样本制备的质量与一致性，包括处理那些极具挑战性的组织/生物流体（如淀粉质储藏根和哺乳动物血浆），且不受样本中RNA污染物或RNA降解状态的影响。我们将用于sRNA分离的TraPR方案与基于总RNA的标准sRNA文库制备流程进行了对比，所用源组织涵盖拟南芥花芽、果蝇卵巢、小鼠肝脏及血浆等多种样本。