Next-generation deep sequencing (NGS) technology represents a powerful and innovative approach to profile small RNA. Currently, there are a number of large-scale and benchtop sequencing platforms available on the market. Although each platform is relatively straightforward to operate, constructing cDNA libraries can be the most difficult part of the NGS workflow. Constructing quality libraries is essential to obtaining a successful sequencing run of high-quality reads and coverage. The quality and yield of RNA affect hybridization and ligation of sequencing adapters. In the field of biomarker discovery, there has been an interest in profiling exosomal RNA from biological fluids. However, very little RNA yield is obtained when extracting RNA from exosomes, thus making library construction difficult. Here, researchers from La Trobe University describe an optimized protocol for constructing small RNA libraries from low yields of RNA, in particular, extracted from exosomes isolated from biological fluids.
Small RNA profiles of exosomes secreted from various biological fluids
Exosomes were isolated from 1 ml of plasma and serum, 20 ml of urine, and 5 ml of synovial fluid using sequential ultracentrifugation (UC). Samples were concentrated on a SpeedVac® instrument to 6 μl. RNA profiles were analyzed using a small RNA assay kit and ran on an Agilent® Bioanalyzer® Instrument. A ladder was run during each Bioanalyzer® run which was used to calibrate the fluorescence units (FUs), nucleotide (nt) size, and RNA yield which may differ from chip to chip. These samples did not contain detectable large RNA species (≥200 nt) as examined on a RNA Pico Bioanalyzer® chip. Therefore, the small RNA and miRNA (10–40 nt) concentration was used to calculate percentage of miRNA and miRNA yield (ng)