Quantitative real-time PCR

Total RNA was isolated using an RNeasy Mini Kit (Qiagen, Valencia, CA). Briefly, cDNA was synthesized using a high capacity cDNA reverse transcription kit (Applied Biosystems). Primers specific for each of the signaling molecules were designed using NCBI/Primer-BLAST and used to generate the PCR products. For the quantification of gene amplification, Real-time PCR was performed using an ABI 7300 Sequence Detection System in the presence of SYBR-Green. The sequence of gene-specific primers are given in Supplementary Information. Target sequences were amplified at 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. HK-GAPD was used as endogenous normalization control. All assays were performed in triplicate and were calculated on the basis of ΔΔCt method. The fold change in mRNAs expression was determined according to the method of 2^−ΔΔCT.