Quantifying Gene Expression using qPCR

Quantitative PCR (qPCR) reactions were performed using PowerUp SYBR Green Master Mix (Applied Biosystems). Each qPCR reaction contained 10 ng of cDNA as template, 300 nM of each primer (Table 2) and 1x of PowerUp SYBR Green Master Mix; in a final volume of 10 µL adjusted with nuclease-free water (Thermo fisher). The PCR program consisted of 2 min 50°C for UDG activation, 2 min 95°C for Dual-Lock DNA polymerase followed by 40 cycles at 95°C for 15 sec and 60°C for 1 min. Primers for each target gene were designed using Primer3 web (Untergasser et al., 2012 (link)). Each qPCR reaction was performed in duplicates with a negative control in each run using an Applied Biosystems ABI 7500 real-time PCR thermocycler (Applied Biosystems). The amplification specificity of each PCR product was monitored using the melting curve analysis in Sequence detection system (SDS) version 1.4.0.27 (Applied Biosystems) and visualizing the PCR products in a 2% agarose gel. The relative gene expression was estimated by the 2^-ΔΔCT method (Livak and Schmittgen, 2001 (link)), using β-actin as a reference gene (GenBank XM_026821238.1) as was previously described (Hajeri et al., 2014 (link)).