Total fluid RNA was extracted using miRCURY RNA Isolation Kit for Biofluids (Exiqon). Exosomes were isolated using miRCURY Exosome Isolation Kit (Exiqon), and total RNA (both cellular and exosomal) was isolated using miRCURY RNA isolation kit for cells and plants (Exiqon). Non-exosomal RNA was isolated from the medium after exosome isolation using miRCURY RNA Isolation Kit for Biofluids (Exiqon).
miRNAs were reverse transcribed using miRCURY LNA Universal RT miRNA PCR, polyadenylation and cDNA synthesis kit (Exiqon) and mRNA with Transcriptor First Strand cDNA Synthesis Kit (Roche). cDNA templates were assayed in 10μl PCR reactions with either LightCycler 96 System or LightCycler 480 Real-Time PCR System (Roche) according to the protocol of miRCURY LNA Universal RT miRNA PCR for miRNA samples and the protocol of Fast Start Universal Probe Master (Rox) (Roche) for mRNA samples. miRNA levels were determined using hsa-miR-106b-5p (205884, Exiqon), -3p (204020, Exiqon), hsa-miR-93-5p (204715, Exiqon), -3p (204470, Exiqon), hsa-miR-25-5p (204031, Exiqon), -3p (204361, Exiqon) LNA™ PCR primer sets. mRNA levels were determined using Universal Probe Library System (Roche, specific primer-probe pairs are listed in Supplemental Material) and Assays-on-Demand target mixtures for MCM7 (Hs00428518, Applied Biosystems) and PPIA (Hs04194521_s1, Applied Biosystems). For analysis, Roche LC Software was used for Cp determination (by the second derivative method) and for melting curve analysis (miRNA samples). GAPDH and PPIA were used for normalisation.
The samples were treated with oxPAPC (30μg/ml) for 4h in EGM or with fresh EGM (control). Total RNA was extracted with TriReagent (Sigma-Aldrich). The profiling and data analysis was executed at Exiqon Services, Denmark. The quality of the total RNA was verified by an Agilent 2100 Bioanalyzer profile. 200ng total RNA from sample and reference was labelled with Hy3™ and Hy5™ fluorescent label, respectively, using the miRCURY™ LNA Array power labelling kit (Exiqon, Denmark) following the procedure described by the manufacturer. The Hy3™-labeled samples and a Hy5™-labeled reference RNA sample were mixed pair-wise and hybridized to the miRCURY™ LNA array version 11.0 (Exiqon, Denmark), which contained capture probes targeting all miRNAs for human, mouse or rat registered in the miRBASE version 13.0 at the Sanger Institute. The hybridisation was performed according to the miRCURY™ LNA array manual using a Tecan HS4800 hybridisation station (Tecan, Austria). After hybridisation the microarray slides were scanned and stored in an ozone free environment (ozone level below 2.0ppb) in order to prevent potential bleaching of the fluorescent dyes. The miRCURY™ LNA array microarray slides were scanned using the Agilent G2565BA Microarray Scanner System (Agilent Technologies, Inc., USA) and the image analysis was carried out using the ImaGene 8.0 software (BioDiscovery, Inc., USA). The quantified signals were background corrected (Normexp with offset value 10 (19) and normalized using the global Lowess (LOcally WEighted Scatterplot Smoothing) regression algorithm. Full paper
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