Lightcycler 480 cybr green 1 master

cDNA was synthesized with a random primer using 1^st strand cDNA synthesis kit (Roche Diagnostics). Real-time quantitative PCR was performed using a LightCycler 480 instrument (Roche Diagnostics). The 20-μL reaction mixture contained 10 μL of LightCycler 480 CYBR Green I Master (Roche Diagnostics), 0.5 μM of each primer pair (Supplementary Table 4), and 5 μL of 20-fold diluted cDNA. The cycling program steps were as follows: 95 °C for 5 min; 40 cycles of 95 °C for 10 s, 60 °C for 10 s, and 72 °C for 10 s (signal acquisition). The relative abundance was calculated by the delta-delta-Ct method.

For analysis of gene expression in the livers, ∼15 mg of frozen liver tissue was homogenized with TRIzol reagent (Ambion, Cat No. 15596-018) and RNA was extracted following the manufacturer’s protocol. The concentration of extracted RNA was determined using Nanodrop ND-1000 spectrophotometer (Thermo Scientific). First-strand cDNA was synthesized from 2.0 μg of total RNA with Superscript III first-strand synthesis kit (Invitrogen) according to the manufacturer's protocol. For real-time RT-PCR reactions, the cDNA was diluted 15-fold. Sequences of the primers are available by request. Real-time PCR analysis was conducted on Roche LightCycler480 detection system (Roche Diagnostics) with SYBR Green as probe (LightCycler480 CYBR Green I Master, Roche). Relative gene expression levels were calculated using the comparative Ct method by normalization to geometric mean of expression levels of four housekeeping genes (GAPDH, β-actin, m18S, and Hprt1). Unpaired t-test was used to test for statistical significance.

Approximately 100 mg of frozen dorsal skin tissue of WT and DKO mice samples was homogenized in PURzol reagent (Bio-Rad, catalog no. 7326890) and RNA isolated via the Aurum™ Total RNA Fatty and Fibrous Tissue Pack (Bio-Rad, catalog no. 732-6870). The concentration of isolated RNA was determined with a Nanodrop ND-1000 spectrophotometer (Thermo Scientific). ProtoScript® II First Strand cDNA Synthesis Kit (New England BioLabs, catalog no. E6560) was used for cDNA strand synthesis from 1 µg sample RNA. One WT mouse sample was used for several extra reactions simultaneously as a standard for quantification curves. All qPCR was performed with a 15X dilution of cDNA, unless otherwise noted. Curves were calculated with 3X, 9X, 27X, and 81X cDNA dilutions. Real-time PCR analysis was conducted on a Roche LightCycler®480 detection system (Roche Applied Science) with SYBR Green as the probe (LightCycler®480 CYBR Green I Master, Roche Applied Science). Gene expressions were normalized to Gapdh and analyzed as a relative expression of fold-difference from the expression level of WT mice at PD26 of the same sex, using the comparative Ct method. Sequences of primers are available by request.