Megaplex primer pool a v2

Expression of miRNAs was assessed using single miRNA assays for real-time RT-PCR (Applied Biosystems; Rotkreuz, Switzerland). Megaplex Primer Pool A v2.1 (Applied Biosystems) was used for RT-PCR according to manufacturer's instructions. A preamplification step using PreAmp Primer Pool A v.2.1 (Applied Biosystems) was added to increase sensitivity. RNU44 was previously found to be the most stable control miRNA in such experimental setup and was therefore used as a reference miRNA for normalization and relative expression calculations [22 (link)]. Expression of the potential target genes was analyzed with quantitative real-time RT-PCR by using Assay-on-Demand reagents (Applied Biosystems). PUM1 was previously found to be the most stable endogenous control mRNA and was therefore used as a reference gene to normalize expression levels of target mRNAs [22 (link)]. Relative quantitation of all targets was calculated by the comparative cycle threshold method outlined in user bulletin number 2 provided by Applied Biosystems. qPCR probe sequences are described in Supplementary Table 1 (see Supplementary Table 1 in the Supplementary Materials available online at http://dx.doi.org/10.1155/2014/897249).