Quantitative miRNA Expression Profiling

To isolate miRNA-containing RNAs, cells were lysed and homogenized with Acid Phenol (Ambion®, Life Technologies, Darmstadt, Germany) and RNA was isolated using the miRVana RNA isolation kit (Ambion®, Life Technologies, Darmstadt, Germany) according to the instructions provided by the manufacturer. RNA concentration was measured using the Nanodrop 1000 (Thermo Scientific, Waltham, USA) and RNA integrity was assessed using the Bioanalyzer (Agilent, Santa Clara, USA).
cDNA synthesis and primer design (Tab. 2) for the analysis of miRNA expression, profiling by miQPCR was carried out as previously described31 (link). In brief, 10 ng of total RNA was used for cDNA synthesis and a fraction of the synthesized cDNA (equivalent to 1% of final cDNA volume) was used for individual qPCR assays. Primer design was performed as described in31 (link), miRNA specific primers used in this study are listed in Table 2. qPCR assays were performed in a 96 well format and run on a ViiA7 thermocycler (Applied Biosystems®, Life Technologies, Darmstadt, Germany) using Bryt^® Green (Promega, Mannheim, Germany) miRNA amplification parameters were as follows: 95 °C for 10 minutes (1 cycle), 95 °C for 15 seconds and 60 °C for 35 seconds (50 cycles), followed by melting curve analysis. qPCR data were analyzed by qBase software v.1.3.546 (link).