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

Abstract

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) .

Circulating microRNAs as clinically useful biomarkers for Type 2 Diabetes Mellitus: miRNomics from bench to bedside

“…All these factors together contribute to the significant differences between plasma and serum, making the choice of plasma the preferred solution, as reported in multiple studies. 40,83,84 Other important considerations relate to blood collection procedures, as shear forces can induce hemolysis, which has been shown to alter the expression of some miRNAs in plasma. It should be noted that many circulating miRNAs have been associated with hemolysis 85À88 and caution should be taken when selecting any of them as a potential disease biomarker (Supplementary Table 2).…”

“…Another important consideration concerns the blood processing protocol, which should be carefully reviewed as it can be a source of critical variation in the final results. It should be noted that remaining platelets and/or blood cells in plasma samples may cause relevant artefacts in the total amount of miRNAs, 90À93 thus consecutive and appropriate centrifuge steps 40 are strongly required to remove them from plasma and to obtain a platelet-poor-sample. In this regard, a high degree of heterogeneity in the protocols used in our series of studies is evident (Table III).…”

MicroRNA circolanti come biomarcatori per il diabete mellito di tipo 2: avanzamenti e prospettive future

“…-pre-analitiche, quali la scelta del biofluido (plasma o siero) e di raccolta campione (emolisi) [23]; -analitiche, quali la procedura di isolamento dell'RNA, la scelta della metodica e piattaforma di analisi e DELLA strategia di normalizzazione; -fattori individuali correlati al paziente.…”

Non-Coding RNAs: Novel Players in Insulin Resistance and Related Diseases

“…However, use of different profiling platforms and different operative procedures, as well as differences in study population and tissues analyzed, led to inconsistent results and nonreproducible data [ 203 ]. Therefore, standardized samples collection protocols and consistent analytical procedures are strongly needed [ 204 ]. Several miRNAs signatures have been proposed as diagnostic tools for obesity, diabetes and their metabolic complications.…”