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

Summary 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) .