Quantitative Real-Time PCR Protocol

Total RNA was purified using the RNeasy mini kit (Qiagen). Complementary DNA (cDNA) was synthesized in a 50 µL volume using Super-Script™ III First-Strand Synthesis reagents (Seoul, Korea, Invitrogen). The cDNA was amplified using TaqDNA polymerase (Invitrogen, Seoul, Korea) in the presence of 1 μM oligonucleotide primers. The quantitative real-time PCR was performed using the iQ5 real-time system and the iQ SYBR™ Supermix (BioRad, Seoul, Korea). Expression of the target mRNAs relative to housekeeping gene expression (GAPDH mRNA) was calculated using the threshold cycle (C_T) as r = 2^–Δ(ΔCT), where Δ C_T = C_{T target} − C_{T GAPDH} and Δ(Δ C_T) = Δ C_{T D8} − Δ C_{T D0}. The primer sequences are shown in Table 2 [19 (link)].