Relative Gene Expression Analysis by qPCR In Vitro

For analyzing gene activation and protein expression, PANC-1 cells were seeded in duplicate into 24-well plates at a density of 1 × 10⁵ cells/well. TR14 or control aptamer (IRRE) conjugated to C/EBPα-saRNAs was added directly to the cells, at a final concentration of 100 nM. The treatment was repeated 24 h later, and cells were harvested at final incubation times of 72 h. Total RNA was extracted for reverse transcription using an RNeasy kit (QIAGEN). Target cDNA amplification and real-time PCR were performed using a Bio-Rad kit (SsoAdvanced Universal SYBR Green Supermix). For normalization, the reference gene 18S was used.