Icycler iq5 optical system software version 2

Total RNA was isolated using the Aurum^TM Total RNA Mini kit (Bio-Rad Laboratories Srl, Milan, Italy). Following DNAse I treatment, cDNA was synthesized using oligo-dT primers and using ImProm-II^TM Reverse Transcriptase (Promega Corporation, Madison, WI) according to the manufacturer’s instructions. Quantitative real time RT-PCR was performed in a total volume of 15 μl with SYBR Green PCR Master Mix (Bio-Rad Laboratories Srl) and 200 nM concentration of each primer. The primer sequences were as follows: PPARγ sense: 5′-TCAAACGAGAGTCAGCCTTTAACG-3′ and antisense: 5′-AGTGGGAGTGGTCTTCCATTACG-3′; Catalase sense: 5′-TTTCCCAGGAAGATCCTGAC-3 and antisense: 5′-ACCTTGGTGAGATCGAATGG-3′, HO-1 sense: 5′-CCAGCGGGCCAGCAACAAAGTGC-3′ and antisense 5′-AAGCCTTCAGTGCCCACGGTAAGG-3′; GAPDH sense: 5′-TGCACCACCAACTGCTTAGC-3′ and antisense: 5′-GGCATGGACTGTGGTCATGAG-3′. Reactions were carried out in triplicates using the Real-Time Detection System iQ5 (Bio-Rad Laboratories Srl) supplied with iCycler IQ5 optical system software version 2.0 (Bio-Rad Laboratories Srl). Melt curve analysis was performed to confirm the specificity of the amplified products. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an endogenous control.

Total RNA was isolated using an RNeasy Mini kit (Qiagen, Hilden, Germany). Following DNAse I treatment, cDNA was synthesized from 1 µg of total RNA using ImProm-II Reverse Transcriptase (Promega Corporation, Madison, WI) according to the manufacturer's instructions. Real time RT-PCR was performed with SYBR Green PCR Master Mix (Bio-Rad, Hercules, CA) and 200 nM concentration of each primer. Sequences of all primers used are indicated in Table S1. Reactions were carried out in triplicates using the Real-Time Detection System (iQ5 Bio-Rad, Milan, Italy) supplied with iCycler IQ5 optical system software version 2.0 (BioRad). The thermal cycling conditions comprised an initial denaturation step at 95°C for 3 minutes, followed by 40 cycles at 95°C for 10 seconds and 60°C for 30 seconds. Levels of gene expression in each sample were quantified applying the 2^−ΔΔC_T method, using GADPH as an endogenous control.

Total RNA was isolated using the Aurum™ Total RNA Mini kit (Bio-Rad Laboratories Srl, Milan, Italy). Following DNAse I treatment, cDNA was synthesized using oligo-dT primers and using ImProm-II™ Reverse Transcriptase (Promega Corporation, Madison, WI) according to the manufacturer’s instructions. Quantitative real time RT-PCR was performed in a total volume of 15 μl with SYBR Green PCR Master Mix (Bio-Rad Laboratories Srl) and 200 nM concentration of each primer. The primer sequences were as follows: hMC1R sense: 5′-CCTGAAGACCTCACTAGG -3′ and antisense: 5′-CATCTTGTAGAGCCTGAG-3′; mMC1R sense: 5′-CAAGGAGGTGCTGCTGTG-3 and antisense: 5′-TAGACAAATGGAGATCAGGAAGG-3′, β-actin sense: 5′-GACAGGATGCAGAAGGAGATTACT -3′ and antisense 5′-TGATCCACATCTGCTGGAAGGT-3′. Reactions were carried out in triplicate using the Real-Time Detection System iQ5 (Bio-Rad Laboratories Srl) supplied with iCycler IQ5 optical system software version 2.0 (Bio-Rad Laboratories Srl). Melt curve analysis was performed to confirm the specificity of the amplified products. β-actin was used as an endogenous control.