Identifying MiR-140-3p as a stable internal reference to normalize MicroRNA qRT-PCR levels of plasma small extracellular vesicles in lung cancer patients

Exploration of a stably expressed gene as a reference is critical for the accurate evaluation of miRNAs isolated from small extracellular vesicles (sEVs). In this study, we analyzed small RNA sequencing on plasma sEV miRNAs in the training dataset (n = 104) and found that miR-140-3p was the most stably expressed candidate reference for sEV miRNAs. We further demonstrated that miR-140-3p expressed most stably in the validation cohort (n = 46) when compared to two other reference miRNAs, miR-451a and miR-1228-3p, and the commonly-used miRNA reference U6. Finally, we compared the capability of miR-140-3p and U6 as the internal reference for sEV miRNA expression by evaluating key miRNAs expression in lung cancer patients and found that miR-140-3p was more suitable as a sEV miRNA reference gene. Taken together, our data indicated miR-140-3p as a stable internal reference miRNA of plasma sEVs to evaluate miRNA expression profiles in lung cancer patients. •We searched for optimal internal reference for sEV miRNAs in sequencing results.•The candidate internal reference was evaluated for expression using qRT-PCR method.•miR-140-3p is the most stably expressed plasma sEV miRNA in lung cancer patients.•miR-140-3p is a better reference gene than U6 to evaluate miRNA expression profile.