Sybergreen kit

Isolated miRNA was transcribed into cDNA using the miScript RT kit (Qiagen, Hilden, Germany). 5 μl miRNA sample were processed following the manufacturer’s protocol using the HiFlex Buffer. After reverse transcription the samples were diluted in 80μl RNase-free water. qPCR was performed using the miScript primer assays (miR-21, miR-31, miR-520e, miR-17, miR-106b) from Qiagen (Qiagen, Hilden, Germany). A master mix was prepared consisting of miScript Universal primer, miScript Primer assay, SyberGreen kit and RNase-free water (Qiagen, Hilden, Germany) following the manufacturer’s protocol. Samples were analyzed as triplicates in a 364 well plate with the Light Cycler 480 from Roche (Roche, Basel, Switzerland). As reference, miRNA-93 (Qiagen, Hilden, Germany) was used.

For measurement of DPH3 and OXNAD1 expression, reverse transcription reaction was performed using 1 μg of RNA and oligo-dT primers using a cDNA synthesis kit (Thermo Scientific, Waltham, USA). DPH3 and OXNAD1 expression levels were then determined by quantitative real-time PCR using a Syber Green kit (QIAGEN). The real-time PCR was carried out in triplicates in 384-well layouts using QuantiTect primers (QIAGEN) specific for DPH3 (QT00223083), OXNAD1 (QT00074235) and the GUSB (QT00046046), a housekeeping gene used as an internal standard. DPH3 and OXNAD1 expression levels were calculated using GUSB expression as a reference and relative quantification was performed using the ΔΔCT method and log2 transformation. The expression levels were plotted using box plots and statistical differences were determined using two sided t-tests.