2100 bioanalyzer rna system

Total RNA was extracted from each MN sample using ReliaPrep™ RNA Cell Miniprep System kit (Promega) and reverse transcribed using the TaqMan® MicroRNA Reverse Transcription Kit (ThermoFisher Scientific) and First Strand cDNA Synthesis kit (GE Healthcare). Exosomes were collected by ultracentrifugation and assessed by the NanoSight NS300 System (Malvern Panalytical). ex-miRNA extraction was performed with the combination of miRNeasy kit and RNeasy Cleanup Kit (Qiagen) and assessed through the 2100 Bioanalyzer RNA system (Agilent Technologies). Reverse transcription of ex-miRNA was followed by preamplification with TaqMan® Preamp Master Mix kit (ThermoFisher Scientific). Gene and miRNA expression levels were assayed on the 7500 Real Time PCR System (Applied Biosystem). Relative expression quantification was performed by the 2^(-ΔΔCt) method, using 18S or RNU6 as reference. All data are mean of triplicates. Only genes and miRNAs with Ct < 35 were taken into consideration for subsequent analysis. All the TaqMan® assay IDs are available upon request.

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.

Saliva samples (1 mL) were thawed on ice to avoid Extracellular Vesicles’ (EVs) thermal damage. Then, they were centrifuged at 4000× g for 30 min at 4 °C to remove any cell debris and aggregates. Supernatants were ultracentrifuged at 110,000× g for 75 min at 4 °C in order to pellet EVs, then stored at −20 °C.
miRNAs’ isolation from the obtained EVs was performed with the combination of the miRNeasy Kit and RNeasy Cleanup Kit (Qiagen, Hilden, Germany), according to the manufacturer’s protocol. They were eluted in 20 µL of nuclease-free water and stored at −80 °C until used. EV miRNAs’ quality and integrity were assessed through the “2100 Bioanalyzer RNA system” with the Pico Kit (Agilent Technologies, Santa Clara, CA, USA), but the concentration (ng/µL) was assessed by a Quantus Fluorometer (Promega, Milan, Italy).