Quantitative Real-Time PCR Analysis of Gene Expression

RT-PCR analysis for gene expression and the quantitative real-time PCR (qPCR) were performed by an intercalator-based method (Roche Applied Science, Mannheim, Germany), as described previously^{15 (link),19 (link)}. The glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene was used as an internal control. Experiments were performed in duplicate and the results were analyzed by software (LightCycler Software Version 4.0, Roche Applied Science). Expression was detected using the relevant primers for the five selected genes detected by gene microarrays: h-GAPDH forward: 5′-AGG TCA TCC CTG AGC TGA ACG G-3′, reverse: 5′-CGC CTG CTT CAC CAC CTT CTT G-3′; h-BMP8B forward: 5′-CTT TCG TGG TCA CTT TCT TC-3′, reverse: 5′-TGG ACG TCA TCA AAG ATC C-3′; h-CCR6 forward: 5′-GGG AAT CAA TGA ATT TCA GC-3′, reverse: 5′-CAA TCG GTA CAA ATA GCC TG-3′; h-HOXA9 forward: 5′-ATT GGA GGA AAT GAA TGC TG-3′, reverse: 5′-GAA ACC CCA GAT TCA TCA AG-3′; h-NANOG forward: 5′-CCA GAA CCA GAG AAT GAA ATC-3′, reverse: 5′-TGG TGG TAG GAA GAG TAA AG-3′; h-S100A8 forward: 5′-GTA TAT CAG GAA AAA GGG TGC-3′, reverse: 5′-TAC TCT TTG TGG CTT TCT TC-3’.