Upzol

Total RNAs were extracted with phenol-chloroform using Upzol (Dutscher, Brumath, France). Synthesis of cDNA was performed using Maxima First cDNA Synthesis Kit (ThermoFisher Scientific) from 1 μg of total RNA. cDNA (50 ng/µL) was used as a template for quantitative PCR (qPCR), and mixed with primers (200 nM), SYBR™ Green PCR Master Mix (ThermoFisher Scientific) or TaqMan mix (Roche) and Universal Probe Library probes (100 µM) (ThermoFisher Scientific) for the gene of interest. Reactions were performed in triplicate. qPCR analyses were carried out with the FX96 Thermocycler (Biorad, Hercules, USA). Relative mRNA levels were calculated using the Comparative Ct (ΔΔCT) method. Gene expression was normalized with hACTB or mRplp. Primer sequences used are listed in Supplementary Table 1.

RNA was extracted with phenol-chloroform using Upzol (Dutscher, Brumath, France). Synthesis of cDNA was performed using Maxima First cDNA Synthesis Kit (ThermoFisher Scientific) from 1 μg of total RNA. Generated cDNA (50 ng/µL) was used as a template for quantitative PCR (qPCR) run, and mixed with primers (200 nM), SYBR™ Green PCR Master Mix (ThermoFisher Scientific) or TaqMan mix (Roche) and Universal Probe Library probes (100 µM) (ThermoFisher Scientific) for the gene of interest. Reactions were performed in triplicate. qPCR analyses were carried out with the FX96 Thermocycler (Biorad, Hercules, USA). Relative mRNA levels were calculated using the Comparative Ct (ΔΔCT) method. mRNA levels of 2 (Gapdh/Actb) housekeeping genes were used for normalization. Primers sequences and housekeeping genes used are listed in Supplementary Table 1.

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.