Sybr premix ex tag mastermix kit

We employed RNeasy reagent (Qiagen, Germany) to harvest the whole RNA. RT-qPCR was conducted with SYBR Premix ex TAG Mastermix kit (Takara, Japan) on the ICycler real-time system (Bio-Rad Laboratories, USA) as manual described. Glyceraldehyde-3-phosphate dehydrogenase was an internal control. The relative RNA expression was analyzed by the 2^−ΔΔCt approach and presented as the target gene/internal control ratio [2^{−ΔΔCt (target gene-internal control)}] (28 (link)). The data were obtained from three independent experiments in triplicate one time. The primers of CXCR4 are 5’-ACTACACCGAGGAAATGGGCT-3’ (F) and 5’-CCCACAATGCCAGTTAAGAAGA-3’ (R). The primers of GAPDH are 5’-CTGGGCTACACTGAGCACC-3’ (F) and 5’-CTGGGCTACACTGAGCACC-3’ (R).

Total RNA was isolated using RNeasy reagents (Qiagen, Hilden, Germany), and real-time PCR (qRT-PCR) was performed using an iCycler Real-Time System (Bio-Rad Laboratories, Hercules, CA, USA) with a SYBR Premix EX Tag Mastermix kit (TaKaRa, Shiga, Japan) according to the manufacturer’s instructions. Glyceraldehyde-3-phosphate dehydrogenase was used as an internal reference for gene expression. The relative mRNA expression of target genes was expressed using the 2^-ΔΔCt method. Experiments were repeated three times [30 (link)]. The same method was used for mRNA detection in subsequent cell experiments. The primers used are listed in Additional file 2: Table S1.