RNA isolation and real-time RT-PCR

Total RNA was extracted using Trizol reagent (Takara, Dalian, China). A 4 μg template RNA was used to synthesize the first-strand cDNA using a Prime Script RT reagent kit (RR047A TakaraBio, Tokyo, Japan). Quantitative real-time RT-PCR analysis was performed using SYBR^® Fast qPCR Mix (TaKaRa, Shiga, Japan) and a Agilent Technologies Stratagene Mx3000p Real-Time System (Agilent, USA) with SYBR Green to determine the mRNA expression level of a gene of interest. A reference gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), was used to normalize gene expression levels. The mRNA abundance was analyzed using the comparative threshold cycle (2^−ΔΔCT) method. The primers used in the RT-PCR were as follows: CDK5RAP3: forward: 5′-ATTTTTGGCCGATACTCTTCACA-3′; reverse: 5′-TCATAGTTGACATTCCGAACCAG-3′; VEGFA: forward: 5′-CATGAACTTTCTGCTGTCTTGG-3′; reverse: 5′-CATTTGTTGTGCTGTAGGAAGC-3′; GAPDH: forward 5′-AAGGTGAAGGTCGGAGTCAA-3′; reverse 5′-CCATGTAGTTGAGGTCAATGAAGG-3′. All samples were measured with at least three independent experiments and the results are expressed as the mean ± SD of the comparative analysis.