Quantifying SDF-1α and CXCR4 Gene Expression

Total RNA was extracted from the cells using TRIzol® reagent (Invitrogen Life Technologies, Carlsbad, CA, USA) and was reverse transcribed into a total of 1 μl (60 ng/μl) cDNA using a One-Step RT-PCR kit (TransGen Biotech Co., Ltd., Beijing, China), according to the manufacturer’s instructions. Quantification of gene expression was performed using an ABI PRISM 7500 Sequence Detection system (Applied Biosystems Life Technologies, Foster City, CA) with SYBR® Green (TransGen Biotech Co., Ltd.). The relative mRNA expression levels of SDF-1α and CXCR4 were normalized to that of β-actin using the 2^−ΔΔCT method (20 (link)). The following primer sequences were used (Shanghai GenePharma Co., Ltd., Shanghai, China): Rat CXCR4, forward 5′-GCTGAGGAGCATGACAGACA-3′ and reverse 5′-GAT GAAGGCCAGGATGAGAA-3′ (21 (link)); rat SDF-1α, forward 5′-CTGTTGTGCTTACTTGTTTAAGGCTTTGTC-3′ and reverse 5′-GACGCCAAGGTCGTCGGT-3′ (22 (link)) and rat β-actin, forward 5′-GCTACAGCTTCACCACCACA-3′ and reverse 5′-GCCATCTCTTGCTCGAAGTC-3′. The cycling conditions were as follows: 95°C for 10 min, followed by 40 cycles of 95°C for 15 sec and 72°C for 35 sec, for telomere PCR. The experiments were repeated three times with triplicates of each sample.