Abi prism sequence detection software version 2

c-Fos (Hs99999140-m1), CTNNB1/β catenin (Hs003550489-m1) and, as endogenous control, GAPDH (Hs00266705-g1) TaqMan Gene Expression Assays (Applied Biosystems, Foster City, CA) were used. Total RNA (1 μg) was reverse transcribed with oligo d (t) using a Transcriptor First Strand cDNA Synthesis Kit (Roche, Penzberg, Germany). Preliminary experiments were conducted for the endogenous control using the C_t slope method to ensure that the quality of each complementary DNA and the dynamic range of amplifications were comparable [44 (link)]. Real-time PCR was then carried out with 20 ng input complementary DNA, 1 × TaqMan Gene Expression Master Mix and TaqMan Gene Expression Assays on a ABI PRISM 7900 HT thermal cycler (Applied Biosystems). Data were analyzed using ABI PRISM Sequence Detection Software version 2.2.2 (Applied Biosystems). Relative expression was determined on triplicate reactions using the formula 2^−ΔCt, reflecting target gene expression normalized to endogenous control levels [44 (link)].

RNA was extracted from melanoma cell lines by TRIzol (Thermo Fisher Scientific) and cDNA was synthesized from 1 μg of RNA using Transcriptor First Strand cDNA synthesis Kit (Roche, Basel, Switzerland), according to the manufacturer instructions. qPCR was carried out using Taqman Gene Expression Assays 20X (Thermo Fisher Scientific, listed in Supplementary Table S4) and TaqMan Gene Expression Master Mix 2X (Applied Biosystems, Foster City, CA, USA). qPCR was carried out with 20 ng input complementary DNA, 1 × TaqMan Gene Expression Master Mix and TaqMan Gene Expression Assays on an ABI PRISM 7900 HT thermal cycler (Applied Biosystems). Data were analyzed using ABI PRISM Sequence Detection Software version 2.2.2 (Applied Biosystems). Relative expression was determined using the formula 2^−ΔCt, reflecting target gene expression normalized to endogenous control genes levels [10 (link)].