Isolation and Analysis of EV-Derived miRNAs

Isolation of miRNAs from EVs was performed with the combination of miRNeasy kit and RNeasy Cleanup Kit (Qiagen), according to the manufacturer's protocol. EV-miRNAs quality and integrity were assessed through the “2100 Bioanalyzer RNA system” (Agilent Technologies). MiRNAs were eluted in 20 μL of Nuclease-Free Water and stored at −80°C, until to use. MiRNAs reverse transcription (RT) and preamplification reactions, followed by real-time RT-PCR analysis with the QuantStudio™ 12K Flex OpenArray^® Platform (Applied Biosystem), were previously described (20 (link)). Gene Expression Suite Software (Applied Biosystem) was used to process miRNA expression data from the “TaqMan™ OpenArray™ Human MicroRNA panel” (ThermoFisher) analysis.

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Macchi C., Greco M.F., Favero C., Dioni L., Cantone L., Hoxha M., Vigna L., Solazzo G., Corsini A., Banach M., Pesatori A.C., Bollati V, & Ruscica M. (2022). Associations Among PCSK9 Levels, Atherosclerosis-Derived Extracellular Vesicles, and Their miRNA Content in Adults With Obesity. Frontiers in Cardiovascular Medicine, 8, 785250.

Publication 2021

miRNeasy kit RNeasy Cleanup Kit Gene Expression Suite Software TaqMan™ OpenArray™ Human MicroRNA panel

Gene expression Human Isolation Microrna Real time pcr analysis Reverse transcription

Corresponding Organization : University of Milan

Other organizations : Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, MultiMedica, Istituti di Ricovero e Cura a Carattere Scientifico, University of Zielona Góra, Medical University of Lodz

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Variable analysis

independent variables

Isolation of miRNAs from EVs was performed with the combination of miRNeasy kit and RNeasy Cleanup Kit (Qiagen), according to the manufacturer's protocol.

dependent variables

EV-miRNAs quality and integrity were assessed through the "2100 Bioanalyzer RNA system" (Agilent Technologies).
MiRNAs reverse transcription (RT) and preamplification reactions, followed by real-time RT-PCR analysis with the QuantStudio™ 12K Flex OpenArray® Platform (Applied Biosystem), were previously described.

control variables

MiRNAs were eluted in 20 μL of Nuclease-Free Water and stored at −80°C, until to use.

controls

No positive or negative controls were explicitly mentioned in the provided information.

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