Fluidigm real time pcr analysis

RNA isolated from fibroblasts was reversely transcribed to complementary DNA, and the expression of dermal ECM genes was analyzed with a quantitative polymerase chain reaction system (BioMark HD, Fluidigm, San Francisco, CA, USA). The threshold cycle (Ct) value of gene expression was calculated by software (Fluidigm Real-Time PCR Analysis, Fluidigm). Biological variation between samples was normalized with the Ct value of the glyceraldehyde 3-phosphate dehydrogenase gene (GAPDH) as a housekeeping gene. Experimental variation between culture plates was normalized on each plate with the Ct value of the control sample, which was treated with 10% FBS, and the relative value calculated by the ΔΔCt method was used for the analysis. To examine the effect of blood plasma or circulating factors on the dermis, the dermal ECM genes shown in Table 2 were quantified. The primers and probes of TaqMan^® Gene Expression Assay (Thermo Fisher Scientific, Waltham, MA, USA) shown in Supplementary Table 2 were used for the analysis.

Gene expression analyses via high-throughput RT-qPCR were performed via Fluidigm dynamic arrays on the BioMark system as described previously [23 (link)]. Data were processed using Fluidigm Real-Time PCR Analysis as well as GenEx software, Version 5.3.6. For normalization, five reference genes (ACT, B2M, GAPDH, GUSB, and HPRT1) were used. Expression changes of the respective genes were displayed as log2-fold change compared to untreated controls by calculating relative quantities corresponding to the ΔΔCq method [55 (link)].