Ten nanograms of total RNA were reverse transcribed using Taqman microRNA Reverse Transcription Kit (Life Technologies, Carlsbad, CA, USA) according to manufacturer’s instructions, as previously described [51 (link)]. The obtained cDNA was amplified using the following Taqman MicroRNA assays: hsa-miR-22, hsa-miR-24, hsa-miR-99a, hsa-miR-194, hsa-miR-214, hsa-miR-335, hsa-miR-339, hsa-miR-708 (Life Technologies, Carlsbad, CA, USA).
To normalize quantitative Real-Time PCR reactions, parallel reactions were run on each sample for RNU48 snRNA. The reactions were performed in triplicate and changes in the target miRNA content relative to RNU48 were determined using the comparative Ct method to calculate changes in Ct, and, ultimately, fold and percent change. An average Ct value for each RNA was obtained for replicate reactions.
To normalize quantitative Real-Time PCR reactions, parallel reactions were run on each sample for RNU48 snRNA. The reactions were performed in triplicate and changes in the target miRNA content relative to RNU48 were determined using the comparative Ct method to calculate changes in Ct, and, ultimately, fold and percent change. An average Ct value for each RNA was obtained for replicate reactions.