Peripheral venous blood samples from the subjects analyzed were processed within 2 h from the collection by centrifugation into EDTA-coated tubes at 2,000 g at 4°C for 20 min. Plasma samples were then stored at −80°C until further analyzed.
Total RNA was extracted from 100 μl of plasma-EDTA using the total RNA Purification kit from Norgen Biotek Corporation (Thorold, ON, Canada) according to the manufacturer’s guidelines. Circulating miRNA levels were analyzed as described in Olivieri et al. (2014 (link)) using a modified real-time approach with the TaqMan miRNA reverse transcription kit and the miRNA assay from Applied Biosystems (Foster City, CA, USA). qPCR reactions were performed on a RotorGene Q HRM instrument (Qiagen, Germany). The plasma levels of circulating miRNAs are reported as relative expression (RE) normalized to the mean of spiked-in synthetic non-human cel-miR-39. The relative expression of each miRNA was reported as 2−ΔCt, with ΔCt being the difference between the Cts of a specific miRNA and those of the cel-miR-39. Cel-miR-39 was used also for the assessment of miRNA recovery from biological samples. Only those samples with a cel-miR-39 recovery higher than 95% were processed for inflamma-miRNA quantification. Each reaction was performed in duplicate.
Total RNA was extracted from 100 μl of plasma-EDTA using the total RNA Purification kit from Norgen Biotek Corporation (Thorold, ON, Canada) according to the manufacturer’s guidelines. Circulating miRNA levels were analyzed as described in Olivieri et al. (2014 (link)) using a modified real-time approach with the TaqMan miRNA reverse transcription kit and the miRNA assay from Applied Biosystems (Foster City, CA, USA). qPCR reactions were performed on a RotorGene Q HRM instrument (Qiagen, Germany). The plasma levels of circulating miRNAs are reported as relative expression (RE) normalized to the mean of spiked-in synthetic non-human cel-miR-39. The relative expression of each miRNA was reported as 2−ΔCt, with ΔCt being the difference between the Cts of a specific miRNA and those of the cel-miR-39. Cel-miR-39 was used also for the assessment of miRNA recovery from biological samples. Only those samples with a cel-miR-39 recovery higher than 95% were processed for inflamma-miRNA quantification. Each reaction was performed in duplicate.
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