Evaluating COX-2 mRNA Expression

In order to evaluate the effect of the drugs on COX-2 mRNA expression, the cells were treated with trabectedin, propranolol, and their combined treatment for 24 h in the presence of 10 µM of NE. Then, the treated cells were harvested, total RNA was extracted using TRIzol reagent (Thermo Fisher Scientific, MA, United States), and the concentrations were quantified with the ND8000 Spectrophotometer (NanoDrop Technologies). Then, 500 ng of total RNA was reverse-transcribed using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems™), and quantitative real-time PCR (qPCR) was performed on the StepOnePlus™ Real-Time PCR System (Applied Biosystems™) by using COX-2 and GAPDH-specific primers (Roelofs et al., 2014 (link)) and PowerUp™ SYBR™ Green Master Mix mRNA quantitative real-time polymerase chain reaction Kit (Applied Biosystems™) according to the manufacturer’s instructions. Gene expression levels were quantified by the comparative ΔΔCt method after normalization for the endogenous reference (GAPDH). All the PCR reactions were performed in duplicate three times.