Abi sequence detection software version 2

The quantitative real-time PCR (qRT-PCR) analysis was performed using an ABI7900HT Real-Time PCR system (Applied Biosystems). The TaqMan RT-PCR reagents (the probes and primers) were obtained from Applied Biosystems. Predesigned and optimized Assays-on-Demand kits (Applied Biosystems, GAPDH:Hs00266705_g1, TRPV 1:Hs00218912_m1) were used for the TaqMan analysis. The reaction parameters were as follows: 2 min at 50 °C, 30 min at 60 °C, and 5 min at 95 °C, followed by 45 cycles of 20 s at 94 °C for denaturation, and 1 min at 60 °C for annealing and extension. All of the measurements were conducted in either duplicate or triplicate. ABI sequence-detection software, version 2.0 (Applied Biosystems) was used for data analysis. A relative quantitation was performed using GAPDH as a reference gene and validated using Norm Finder software. Because all of the assays used were optimized for PCR efficiency by the manufacturer, the mRNA-expression levels were determined using the delta-Ct method.

The real-time RT-PCR analysis was conducted using the ABI7900HT machine (Applied Biosystems). All of the TaqMan RT-PCR reagents, including the primers and probes, were purchased from Applied Biosystems. The TaqMan analysis was conducted using predesigned and optimized Assays on Demand (Applied Biosystems). The following assays were used: HIF-1α (ID: Hs00936371_m1), HIF-2α (ID: Hs01026149_m1), OCT4 (ID: Hs03005111_g1), NANOG (ID: Hs02387400_g1), SOX2 (ID: Hs01053049_s1), REX1 (ID: Hs01938187_s1), MIF (ID: Hs00236988_g1), KLF2 (Hs00360439_g1), PPAR γ (ID: Hs01115729_m1), and GAPDH (ID: Hs00266705_g1). The reaction parameters are 2 min at 50 °C hold, 30 min at 60 °C hold, and 5 min at 95 °C hold, and this was followed by 45 cycles of 20 s at 94 °C for a melting, and 1 min at 60 °C for an annealing/extension. All of the measurements were performed in duplicate or triplicate. The results were analyzed using the ABI sequence-detection software version 2.0 (Applied Biosystems). A relative quantitation was conducted using the GAPDH as a reference gene, and it was validated using the NormFinder software. Since all of the used assays were optimized for PCR efficiency by the manufacturer, the mRNA-expression levels were estimated according to the delta-Ct values.