Quantitative miRNA Expression Analysis

For reverse transcription and RT-qPCR, validated primers and probes from TaqMan^® microRNA Assays Kit (Applied Biosystems, Waltham, MA, USA) were used for hsa-miR-16, hsa-miR-21, hsa-miR-19a, hsa-miR-29a, hsa-miR-23a, hsa-miR-145, hsa-miR-203, hsa-miR-155, hsa-miR-210, hsa-miR-31, and hsa-miR-345. cDNA was synthesized for the miRNA of interest starting from 10 ng of extracted RNA using TaqMan^® MicroRNA Reverse Transcription Kit (Applied Biosystems, Waltham, MA, USA) according to the manufacturer’s instructions. RT-qPCR was then carried out in duplicates for each sample using 2x TaqMan^® Universal Master Mix with no Amperase Uracil N-glycosylase (UNG) (Applied Biosystems, Waltham, MA, USA) as previously described [71 (link)]. RT-qPCR was performed using BioRad CFX96 Real Time System, C1000 Thermal Cycler (Hercules, CA, USA). The cycling conditions were 10 min at 95 °C then 40 cycles of: 15 s at 95 °C and 60 s at 60 °C. The fold change of miRNA expression was calculated using the ∆∆Ct equation where miR-16 is endogenous control and compared to healthy controls.