The isolation of the total RNA was carried out from confluent cells using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s protocol. The quantification of the isolated RNA was performed by a UV-ray spectrophotometer (model SmartSpec Plus; BioRad, Hercules, CA, USA). One microgram of RNA was used for the cDNA synthesis using the ImProm-II Reverse Transcription System cDNA synthesis kit (Promega, Madison, WI, USA). The cDNA was PCR amplified with a CFX96 Touch Real-Time PCR Detection System (Thermal Cycler model C1000 Touch; BioRad, Hercules, CA, USA) using SYBR® Green Supermix 1x (Abs 520 nm BioRad; Hercules, CA, USA). The PCR was performed with the first denaturation step at 95 °C for 30 s and then carried out for 40 cycles at 95 °C for 5 s and 60 °C for 30 s. The relative levels of each sample were calculated by the 2−ΔΔCT method [65 (link)] and normalized with GAPDH housekeeping genes.
The gene-specific primers and housekeeping are inTable 3 .
The gene-specific primers and housekeeping are in