Quantifying PDX1 Gene Expression

Total RNA was extracted using Extract RNA reagent (Evrogen, Moscow, Russia) and reverse-transcribed into cDNA using Mint Reverse Transcriptase (Evrogen, Moscow, Russia). qPCR was performed to determine the gene expression levels in the PDX1-expressing and Control cells on a LightCycler480 Real-Time PCR platform (Roche Applied Science, Mannheim, Germany). QPCRmix-HSSYBR was used to determine the relative RNA expression. Primer sequences are shown in Table S1. The HPRT and DDX23 genes were used as an internal control. The PCR reaction conditions were as follows: 1 cycle at 90 °C for 5 min, 40 cycles at 95 °C for 20 s, 60 °C for 20 s and 72 °C for 35 s; and 1 cycle at 95 °C for 5 s, 55 °C for 60 s, and 97 °C for 15 s. The experiments were performed in triplicate for each sample. A relative expression ratio of PDX1 was normalized by using geometric means of the HPRT and DDX23 expression levels. Calculations were performed according to Ganger et al. [19 (link)] for the relative expression ratio.