Quantitative Real-Time PCR for Gene Expression

Total RNA was isolated using an RNA extraction kit (TaKaRa, Japan) and 1000 ng RNA was used to reverse-transcribed into cDNA by the reverse transcription cDNA kit (TaKaRa, Japan). Quantitative real-time PCR was performed with 20 ng of cDNA using the SYBR® Premix ExTaq™ (TaKaRa, Japan). The mix was added to 96-well plate detected by a Biosystems 7900HT fast real-time PCR system (Life Technologies, USA). The real-time PCR cycling condition is 40 cycles of 95°C 5 sec, 60°C 30 sec [17 (link)]. The sequences of the RT-PCR primers (RiboBio, China) were as follows (5ʹ – 3ʹ): LGALS3BP (forward: 5ʹ-AGGTACTTCTACTCCCGAAGGA-3ʹ, reverse: 5ʹ-GGCCACTGCATAGGCATACA-3ʹ) and GAPDH (forward: 5ʹ-GGT GAA GGT CGG TGT GAA CGG ATT T-3ʹ, reverse: 5ʹ-AAT GCC AAA GTT GTC ATG GAT GAC C-3ʹ). The relative mRNA expression was calculated using the 2^−ΔΔCt method and normalized to GAPDH expression.