For quantitative PCR, Taqman Reverse Transcription reagents, primers and probes were purchased from Life Technologies. STAT2, CXCL11, CCL5, IL-17A, and IFNγ mRNA expression was analyzed using Taqman 20X Assays-On-Demand expression assay mix (assay ID: Hs01013123_m1, Hs04187682_g1, Hs00982282_m1, Hs00174383_m1 and Hs00989291_m1, respectively). The probe was a FAM-labeled MGB probe with a non-fluorescent quencher. As housekeeping gene, we used RPLP0. RPLP0 mRNA expression was determined by using Taqman 20X Assays-On-Demand expression assay mix (assay ID: Hs99999902_m1). The probe was a FAM-labeled MGB probe with a non-fluorescent quencher.
PCR mastermix was Platium® qPCR Supermix-UDG (Life Technologies). Each gene was analyzed in triplicates. The real-time PCR machine was a Rotorgene-3000 (Corbett Research, Sydney, Australia). Reactions were run as singleplex. Relative gene expression levels were determined by using the relative standard curve method as outlined in User Bulletin #2 (ABI Prism 7700 sequencing detection system, Life Technologies). Briefly, a standard curve for each gene was made of 4-fold serial dilutions of total RNA from punch biopsies from the skin of psoriatic patients. The curve was then used to calculate relative amounts of target mRNA in the samples.
PCR mastermix was Platium® qPCR Supermix-UDG (Life Technologies). Each gene was analyzed in triplicates. The real-time PCR machine was a Rotorgene-3000 (Corbett Research, Sydney, Australia). Reactions were run as singleplex. Relative gene expression levels were determined by using the relative standard curve method as outlined in User Bulletin #2 (ABI Prism 7700 sequencing detection system, Life Technologies). Briefly, a standard curve for each gene was made of 4-fold serial dilutions of total RNA from punch biopsies from the skin of psoriatic patients. The curve was then used to calculate relative amounts of target mRNA in the samples.