Protocol to analyze circulating small non-coding RNAs by high-throughput RNA sequencing from human plasma samples

The identification and validation of circulating small non-coding RNA (sncRNA) as biomarkers for disease diagnosis, staging, and response to novel therapies is still a compelling challenge. Pre-analytical variables, such as storage temperature or blood hemolysis, and different analytical approaches affect sncRNA stability, detection, and expression, resulting in discrepancies among studies. Here, we report a systematic standardized protocol to reproducibly analyze circulating sncRNAs, employing high-throughput sncRNA sequencing and qRT-PCR validation, from 200 μL of human plasma samples. For details on the use and execution of this protocol, please refer to Ventriglia et al. (2020), Sebastiani et al. (2017), and Dotta et al. (2018).