Supermix with premixed rox dye

Quantitative real-time PCR (TaqMan method) was performed using SuperMix with Premixed ROX dye (Invitrogen), primer sets were designed using the Roche Universal Probe Library Assay Design Centre and purchased from Eurofins MWG Operon (Ebersberg, Germany) and probes from the Roche Universal Probe Library Mouse Set (Roche Applied Science). Samples were assayed in duplicate and run on an ABI 7900HT Fast Real-Time PCR machine using the following conditions: 95°C for 10 minutes then 40 cycles of 95°C for 15 s and 60°C for 1 min. Primer amplification efficiency was validated, and analysis was performed using the relative standard curve method. Data were normalised to Actb and fold-change is expressed as the ratio of expression of each gene of interest in the treated groups against the average of the VC groups. Statistical analysis was performed using GraphPad Prism 7.0. Data are presented as mean ± s.e.m. and statistical comparisons are described in figure legends. Criterion for significance was P < 0.05. Primer pair and probe information is provided in Supplementary Table 1 (see section on supplementary data given at the end of this article).

Quantitative real-time PCR was performed using SuperMix with Premixed ROX dye (Invitrogen), primer sets designed using the Roche Universal Probe Library Assay Design Centre (sequences in Supplemental Table 1) purchased from Eurofins (MWG Operon) and probes from the Roche Universal Probe Library Mouse Set (Roche Applied Science). Samples were assayed in duplicate and run on an ABI 7900HT Fast Real Time PCR machine using the following cycling conditions: 95°C for 10 minutes then 40 cycles of 95°C for 15 seconds and 60°C for 1 minute. Primer amplification efficiency was validated and analysis was performed using the relative standard curve method. Data were normalized to 18S and fold-change is expressed as the ratio of expression of each gene of interest in the treated groups vs the average of the VC groups (one for the 24-h and one for the 7-d time point, respectively). Statistical analysis was performed using GraphPad Prism 6.0. Data are presented as mean ± SEM. Student t test was used for comparison between VC and DHT groups. Criterion for significance was P < .05. Primer pair and probe information is provided in Supplemental Table 1.