Complete, streamlined, reduced-bias workflow for RNA extraction and small RNA library preparation from serum and plasma samples

The profiling of small RNAs, miRNAs in particular, in human plasma and
serum samples is a rapidly expanding fi eld, largely due to the great promise of
cell-free RNAs as biomarkers. Here we describe a workfl ow using a phenolic
extraction reagent along with magnetic bead-based cleanup for isolation of
total RNA from human serum and plasma, followed by reduced bias small
RNA NGS library preparation. We have developed a protocol using a phenolic
extraction reagent combined with a magnetic bead purifi cation for isolation of
total RNA, including small RNA, from up to 600 μL of plasma or serum in a microcentrifuge tube format. The use of magnetic bead-based purifi cation greatly increases throughput, time to complete, and user-friendliness by avoiding time consuming and tedious alcohol precipitation or column-based cleanups. Isolated RNA is then used as input into a reduced-bias library preparation protocol that uses the proven technology of adapters with randomized ends to reduce ligase bias. We also describe a protocol modifi cation that greatly reduces the number of reads mapping to tRNA and YRNA fragments, which can often be a substantial number of reads in libraries created from serum or plasma RNA. This modifi cation results in a greater number of reads mapping
to miRNAs which, in combination with bias reduction, allows for
detection/discovery of more miRNAs at equal sequencing depth or the ability to sequence samples less deeply than other popular protocols while obtaining comparable data. Here we describe a streamlined, user-friendly workfl ow for isolation of total RNA and generation of reduced-bias small RNA sequencing libraries from plasma and serum samples. This tool will be valuable to the growing number of researchers exploring extracellular small RNA for various reasons, including biomarker discovery.