Human panel 1 2

Cell line RNA and EV RNA was collected using miRCURY RNA Isolation Kit (Exiqon). Seven hundred and forty-two miRNAs were measured using microRNA Ready-to-Use PCR, Human panel I+II (Exiqon) on a Viia 7 Real-Time PCR System (Thermo Fisher). Cell line and EV profiles were normalized to global mean and fold-change analysis was used to compare matched cellular and EV miRNA profiles [36 (link)]. We subsequently validated EV miRNA candidates using a TaqMan Assay normalized to input and cel-miR-39-3p miRNA spike in to account for PCR efficiencies.

miRNA analysis was performed on an independent formalin-fixed, paraffin-embedded (FFPE) sample set including CRC (n = 3), adenomas (n = 3) and NAT (n = 3) samples. miRNA isolation was performed with the High Pure miRNA kit (Roche) and the expression of approximate 800 miRNA were assessed on Human Panel I + II (Exiqon) with the miRCURY^TM Universal RT microRNA PCR protocol according to the manufacturer’s instructions. Normalisation of raw Ct data was performed with interplate calibrators followed by miR-423-5p, as a housekeeping gene expressed at relatively constant levels in our analysed samples. In silico miRNA prediction was performed for all analysed genes using the miRWALK database prediction algorithm including validated mRNA targets [19 (link)] in order to select experimentally verified miRNA interaction information associated with genes, pathways, organs, diseases, cell lines, OMIM disorders, and literature on miRNAs. Subsequently, expression of selected miRNAs in normal, adenoma and cancer samples was compared.

MDA-MB-231 cells were treated ±10 ng/ml VEGFA for 7 days followed by Ready-to-Use PCR microRNA array, Human panel I+II in 384-well plates from Exiqon (Woburn, MA, USA). miRCURY LNA miR-452 antagomir and miRCURY LNA miRNA antagomir control were purchased from Exiqon (Woburn, MA, USA) and transduced per manufacturer.