Fig. 1
Amplification cycles of (A) endogenous sEV-miRNA-151a, B endogenous sEV-miRNA-16 and (C) miR-451a measured by qRT-PCR in 172 samples from blood and ascitic fluid from cancer patients and donors. We observed the lowest variation in cycle amplification when analyzing miR-151a compared to miR-16 within the different tumor types assessed and type of samples collected (SD: 1.66 cycles; CV: 0.056, versus SD: 1.91 cycles; CV: 0.080), finding nearly four fewer cycles of variation in miR-151a amplification (8.5 versus 12.3 cycles) (Fig. 1A, B). Compared to miR-451a amplification, which lacks normalizing features, these differences are three times higher (8.5 versus 19.3 cycles) (Fig. 1A and C). A1-A51 (51 advanced stage NSCLC), L1-L28 (28 early-stage NSCLC), OV1-35 (ovarian cancer patients; AF: Ascitic fluid; PL: Plasma), GB 1–12 (glioblastoma patients) and C1-13 (Healthy donors). D Distance to the mean (DM) was calculated using the normalized values in terms of absolute values. Mean CT normalization of each miRNA in each patient was calculated using the triplicate CT values obtained by qRT-PCR from each sample, that was normalized against the mean value of all miRNA analyzed in the total samples evaluated in the assay. The distance to the mean of all miR-151a individual values obtained from each sample was statistically significant with respect to miR-16 (p < 0.001). E SD: Standard deviation of the mean value of the amplification cycle and CV: coefficient of variation (SD/mean) calculated related to the amplification cycle of miR-151a and miR-16 for each individual group of plasma samples. The coefficient of variation of the control samples is almost doubled in the case of miR-16 amplification (SD: 1.43 cycles; CV: 0.06 versus SD: 0.96 cycles; CV: 0.033). F SD, CV and DM values for plasma and ascetic fluid from ovarian cancer patients regarding miR-16 and 151a levels. miR-151a values remained closer to the overall mean (DM) than those of the miR-16 in the ovarian cancer plasma samples (p = 0.007) and, although this difference was not found when analyzing the ascitic fluid (p = 0.851). G miR-451 normalized levels with miR-151a or miR-16. A moderate correlation rate, primarily associated with those samples with the highest values of miR-451a, was maintained in this extended cohort when its levels were normalized by miR-151a or miR-16 (R.2 = 0.732), R: Correlation coefficient. Spearman’s nonparametric correlation test. H Violin plots illustrate the distribution of log2 fold change normalized miR-451a levels. The calibration against miR-151a would provide clear differentiation between two groups of patients behaving differently from the control value, with values above the 75th or below the 25th percentiles. Red dotted lines represent the median and quartiles. ***p < 0.01 and **p < 0.01