Single-stranded cDNA was synthesized from total RNA using the TaqMan® MicroRNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA). Following reverse transcription, quantitative RT-PCR (qRT-PCR) was performed in duplicate, using TaqMan® MicroRNA Assays (Applied Biosystems) following manufacturer’s instructions. Assays used were: hsa-miR-7 (P/N: 4427975, ID: 000268), hsa-let-7g (P/N: 4427975, ID: 002282), hsa-miR-152 (P/N: 4427975, ID: 000475), hsa-miR-15b (P/N: 4427975, ID: 000390), hsa-miR-301a (P/N: 4427975, ID: 000528), hsa-miR-369-3p (P/N: 4427975, ID: 000557), RNU44 (P/N: 4427975, ID: 001094), and RNU6B (P/N: 4427975, ID: 001093). qRT-PCR reactions were carried out in a 7900HT Fast Real-Time PCR System and data collected using SDS software (Applied Biosystems). qRT-PCR results (absolute Ct data) were imported into GenExsoftware (MultiD Analyses AB, Gothenburg, Sweden). Data assembled from multiple experimental plates was subjected to normalization to common reference samples, autoscaling, and normalization to the average of two endogenous control genes (SNORD44 and U6BsnRNA). Kolomogorov-Smirnov test was performed to assess normality of the data and t-test to assess statistical significance.
