qRT-PCR Analysis of ATF3 Expression

TRIzol reagent (Ambion, CA, USA) was used to isolate total RNA. RNA reverse transcription and RT-qPCR were performed as previously depicted [14 (link)]. cDNA was reverse transcribed from 1 μg of total RNA using a reverse transcription kit (Takara, Japan). β-actin was used as an internal reference. Amplification was performed according to the instructions of 2 × SYBR Green Master Mix (Promega, USA). PCR was performed with an initial denaturation at 95°C for 30 s, followed by denaturation at 95°C for 30 s and annealing/extension at 60°C for 30 s for 40 cycles. Relative gene expression levels were calculated using the 2-ΔΔCt method of qRT-PCR. The primers from NCBI were listed as follows:

ATF3 forward: 5′-TGCTCAGAGAAGTCGGAAGAA-3′;

ATF3 reverse: 5′-TGGCACAAAGTTCATAGGGCA-3′;

GAPDH forward: 5′-AGGTCGGTGTGAACGGATTTG-3′;

GAPDH reverse: 5′-GGGGTCGTTGATGGCAACA-3′.