Taqman gene expression assays primers probe

NucleoZOL (Macherey‐Nagel) was used to perform total RNA extraction according to the manufacturer's recommendations. The First‐Strand cDNA Synthesis Kit (GE Healthcare, Chalfont St Giles, UK) was used to synthesize cDNA from 2 μg of total RNA, according to the manufacturer's instructions. The RT reaction mixture was diluted 1:4 and used as a cDNA template for qPCR. TaqMan quantitative PCR was carried out on a FX96 Thermocycler (Bio‐Rad). The PCR mixture contained TaqMan mix (Roche), 200 nM of primers (see Table S2 for their sequence), Universal Probe Library probe (100 μM, ThermoFisher Scientific) for the gene of interest (TaqMan Gene Expression Assays [Primers/probe], Life technologies), and 50 ng/μg of cDNA template. Reactions were performed in triplicate with the following program: 95°C 10 min, followed with 40 cycles of 95°C for 10 s, 59°C for 30 s. The relative amount of mRNA was calculated using the comparative Ct (ΔΔCT) method, following data normalization against GAPDH housekeeping gene.

RNA was extracted with phenol–chloroform using Upzol (Dutscher, Brumath, France). The Maxima First cDNA Synthesis Kit (Life Technologies) was used to synthesize cDNA from 1 μg of total RNA. The reverse transcription (RT) reaction mixture was diluted 1/20 and used as cDNA template for quantitative PCR (qPCR) analysis. TaqMan qPCR analyses were carried out on a FX96 Thermocycler (Bio‐Rad, Hercules, USA). The PCR mixture contained TaqMan mix (Roche, Boulogne‐Billancourt, France), 200 nM of primers, the Universal Probe Library probe (100 µM) for the gene of interest (TaqMan Gene Expression Assays [Primers/probe]; Life technologies), and 1.67 μl cDNA template. Reactions were performed in triplicate. The relative amount of mRNA was calculated using the comparative Ct (ΔΔCT) method, following data normalization against ACTB for housekeeping genes. The PCR primers used for the qPCR are listed in Supporting Information Table S2.

TRI Reagent (Sigma‐Aldrich) was used to perform phenol–chloroform extractions of total RNA. The First‐Strand cDNA Synthesis Kit (GE Healthcare, Chalfont St Giles, UK) was used to synthesize cDNA from 2 μg of total RNA, according to the manufacturer's instructions. The RT reaction mixture was diluted 1/20 and used as a cDNA template for the qPCR. A TaqMan quantitative PCR was carried out on a FX96 Thermocycler (Bio‐Rad). The PCR mixture contained TaqMan mix (Roche), 200 nM of primers, the Universal Probe Library probe (100 μM) for the gene of interest (TaqMan Gene Expression Assays (Primers/probe), Life technologies) added up with 1.67 μl of cDNA template. All of the reactions were performed in triplicate. The relative amount of mRNA was calculated using the comparative Ct (ΔΔCT) method, following data normalization against 1 housekeeping gene. The PCR primers used for the qPCR are available upon request.