Lightcycler 480 2 real time pcr

Quantitative PCR was performed using SYBR Green I real-time PCR method (Roche, LightCycler® 480 SYBR® Green I Master). The Ct values, as defined by the default setting, were measured using a LightCycler® 480 II realtime PCR (Roche) using a thermal profile of 10 min at 95°C followed by 45 cycles of 10 s at 95°C, 10 s at 60°C and 10 s at 72°C. Single-product amplification was confirmed by post-reaction dissociation analysis. PCR primers were designed with the aid of LightCycler® Probe Design Software 2.0 (v1.0) (Roche). Primer sequences (5’ to 3’) are listed in Supplementary Table 1. Relative gene expression was calculated as the 2^−ΔCt, where ΔCt was determined by subtracting the average Rp49 Ct value from that for each gene.

Formalin-fixed paraffin-embedded (FFPE) skin tissue sections were deparaffinized with 2× histosol baths to remove paraffin followed by 2× ethanol, 100 °C washes which are miscible with histosol and 2 ethanol 95 °C baths (3 minutes for each bath). For rehydration, tissues were incubated in a distilled water bath for 2 h before RNA extraction was preformed. To achieve a high RNA yield per sample, 2 sections from each skin sample and 2 time points were combined from a total number of 3 mice from each mouse type (e.g WT or IL-1α KO and time 0 or 18 h after UV exposure). Total RNA was extracted using the RNeasy FFPT kit (QIAGEN) and 500 ng of total RNA was used for cDNA synthesis using the RevertAid H Minus First Strand cDNA Synthesis Kit (Thermo Scientific). qPCR reactions were all preformed with LightCycler 480 II real-time PCR (Roche). All qPCR primers were previously described in Kim et al. 201329 (link). Data were normalized to the expression of the housekeeping gene β-actin.