Quantifying mRNA Expression in MSCs and HUVECs

Total RNA from MSCs and HUVECs was extracted following manufacturer’s instructions in the RNeasy mini kit (Qiagen, Valencia, CA, USA). Quantitative real-time PCR (RT-PCR) was carried out using an iQ SYBR Super-mix (Bio-Rad, Hercules, CA, USA) on an iQ5 real-time system [28 (link)]. In brief, cDNA was synthesized using SuperScript^™ III First-Strand Synthesis for RT-PCR (Invitrogen, Carlsbad, CA, USA) and amplified using Taq DNA polymerase in the presence of primers (Table 1). The expression of target mRNA relative to Glycer-aldehyde-3-phosphate dehydrogenase (GAPDH) was calculated based on the threshold cycle (C_T) as r = 2^{−Δ (ΔCT)}, where ΔC_T = C_T target − C_{T GAPDH} and Δ(ΔC_T) = ΔC_{T experimental} − ΔC_{T control}.