The gene transcripts encoding six RBPs identified in exosomes were silenced in the cytoplasm by siRNA and the subsequent effect on the amount of esRNA was assessed.

<p>A significant reduction of total RNA in exosomes (esRNA) was shown by post-transcriptional silencing of MVP. <b>(A)</b> Confirmation of gene silencing (silenced transcripts) in the cells after transfection with the siRNAs against the transcripts of HSP90AB1, XPO5, hnRNPH1, hnRNPM, hnRNPA2B1, and MVP with respect to the Negative Control siRNA (scrambled siRNA). Quantitative PCR was performed using cDNA, Fast SYBR Green Master Mix, and gene-specific primers. Gene expression was normalized to that of the housekeeping gene <i>GAPDH</i>. The error bars represent average of 4 biological replicates for hnRNPA2B1 and MVP, and 2 biological replicates for other four transcripts, and the average fold change values presented as RQ (relative quantification). <b>(B)</b> Quantification (the percentage-change, %) of total RNA present in exosomes (ng total esRNA / μg total exosomal proteins) after each gene silencing, <i>i</i>.<i>e</i>. silencing of HSP90AB1, XPO5, hnRNPH1, hnRNPM, and hnRNPA2B1. The silencing of hnRNPA2B1 caused a slight reduction of total RNA present in exosomes, but at non-significant level ≈13%. However, after silencing of hnRNPH1 the amount of RNA in exosomes was increased as compared to negative control. The experiment was performed in duplicates, except from hnRNPA2B1 that was performed in 4 replicates. The graph shows the range of values and median (grey). <b>(C-D)</b> Quantification of total RNA (ng total RNA / μg total protein) present in cells and exosomes after gene silencing of MVP. <b>(C)</b> A slight increase (but not significant, p-value 0.57) of the amount of total RNA in cytoplasm was observed. <b>(D)</b> On the contrary, down regulation of MVP (silencing) caused a significant reduction of the total RNA present in exosomes (esRNA), approximately by 50% (p-value 0.02). The experiment for MVP silencing was performed in four replicates; the graph shows the range of values and median (grey).