Myiq thermocycler

Total RNA from the skin samples was extracted using a Trizol reagent (Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s protocol. cDNA was synthesized from 1 µg RNA using iScript cDNA synthesis kit (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s instructions. Using iQ^TM SYBR Green Master Mix (Bio-Rad, Hercules, CA, USA), cDNA was amplified by a real-time PCR with a Bio-Rad MyiQ thermocycler and SYBR Green detection system (Bio-Rad, Hercules, CA, USA). The standard PCR conditions were 95 °C for 10 min and then 40 cycles at 95 °C for 30 s, 60 °C for 30 s, and 72 °C for 30 s. The expression of XPA, IFNα, IFNβ, IRF1, IRF4, IRF7 gene (Table 1) was normalized to the expression level of the GAPDH mRNA in each sample (23, 40, 44–50). For mRNA analysis, the calculations for determining the relative level of gene expression were made using the cycle threshold (C_t) method. The mean C_t values from duplicate measurements were used to calculate the expression of the target gene with normalization to a housekeeping gene used as internal control and using the formulae 2^−ΔΔCT.

Genomic DNA was extracted for genotyping using PCR, as previously reported (Edwards et al., 1991 ). Total RNA was prepared with TRIZOL reagent (Invitrogen, Carlsbad, CA, USA) and TURBO DNA-free Kit (Applied Biosystems, Foster City, CA, USA). cDNA synthesis was performed with Ready-To-Go Kit (GE Healthcare Life Sciences, Stockholm, Sweden) and analyzed by reverse transcription PCR (RT-PCR). PCR primers were designed by Primer3². Quantitative real-time PCRs (qPCRs) with iQ SYBR Green Supermix (Bio-Rad, Techview, Singapore) were performed by using MyiQ thermocycler and iQ5 optical system (Bio-Rad). qPCR data were normalized to the internal control of PP2AA3 (PP2A) (Czechowski et al., 2005 (link)).

We quantified total AGF load in the 11 tortoise samples and compared it to the AGF load in 10 cattle, 10 goats, 10 sheep, and 10 horses (sample names in red text in Dataset S2A) using quantitative PCR. The same primer pair (AGF-LSU-EnvS and AGF-LSU-EnvS Rev) used in the amplicon-based diversity survey described above was also used for qPCR quantification. The 25-μl PCR reaction volume contained 1 μl of extracted DNA, 0.3 μM of primers AGF-LSU-EnvS primer pair, and SYBR GreenER™ qPCR SuperMix for iCycler™ (ThermoFisher, Waltham, Massachusetts, USA). Reactions were run on a MyiQ thermocycler (Bio-Rad Laboratories, Hercules, California, USA). The reactions were heated at 95 °C for 8.5 min, followed by 40 cycles, with one cycle consisting of 15 s at 95 °C and 1 min at 55 °C. A pCR 4-TOPO or pCR-XL-2-TOPO plasmid (ThermoFisher, Waltham, Massachusetts, USA) containing an insert spanning ITS1-5.8S rRNA-ITS2-D1/D2 region of 28S rRNA from a pure culture strain was used as a positive control, as well as to generate a standard curve. The efficiency of the amplification of standards (E) was calculated from the slope of the standard curve and was found to be 0.89.

Total RNA of MDA-MB-435s, MDA-MB-231 or MFC-7 cells were extracted according to standard protocols with NucleoSpin^®RNA II kit (Macherey-Nagel, Hoerdt, France), and reverse transcribed using RevertAid First Strand cDNA Synthesis Kit (Fermentas/Thermo Scientific). Gene quantifications were performed on 50 ng of cDNA using SYBR^® Premix Ex Taq^™ II (Tli RNaseH Plus, TAKARA/Ozyme) using MyiQ thermocycler (Bio-Rad, Marnes-la-Coquette France). Primers (all from Sigma-Aldrich) were used at final concentration of 200 nM and temperature protocol was 10min start at 95°C, followed by 40 cycles of 15 s at 95°C, 30 s at 57°C and 30 s at 72°C. Relative gene expression levels were normalized to HPRT1 and calculated using the 2^−DDCt method. Primers are listed in Supplementary Table S3.