Powerup sybr green master mix mrna quantitative real time polymerase chain reaction kit

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.

To evaluate the effect of XAV-939 on mRNA expression of β-catenin, PDAC cells were treated with the drug (10 μM) for 48 h. The treated cells were then harvested, total RNA was extracted by means of QIAzol^® Lysis Reagent (QIAGEN Sciences, Maryland 20874, USA), and used for synthesis of cDNA with High Capacity cDNA Reverse Transcription Kit (Applied Biosystems™, Thermo Fischer Scientific Baltics UAB-Lithuania, Vilnius, Lithuania). The relative expression of β-catenin was measured in a StepOnePlus™ Real-Time PCR System (Applied Biosystems™-USA) using specific primers for the gene and PowerUp™ SYBR™ Green Master Mix mRNA quantitative real-time polymerase chain reaction Kit (Applied Biosystems™-Austin, TX, USA). Forward (FW) and reverse (RV) specific primer sequences for β-catenin gene are 5′ GCT GGG ACC TTG CAT AAC CT 3′ and 5′ AAG CAT TTT CAC CAG GGC AG 3′, respectively. Gene expression was normalized to the level of GAPDH within each sample using the relative ΔΔCT method.