Biorad iscript select cdna synthesis kit

One μg of RNA was reverse transcribed using the BIORAD iScript Select cDNA synthesis Kit (Bio-Rad). The resulting cDNA was then amplified by qPCR using iSC SYBR green supermix in the iCycler iQ instrument (Bio-Rad) according to the manufacturer’s instructions. The primers used for this study are listed in Table S4. These experiments were performed in triplicate from three independent RNA extractions. We considered a significant modification in gene expression when the fold changes were greater than 2 or less than -2 with a P value < 0.05.

RNAs were extracted from cells harvested at late-exponential phase and at the same OD of 1. Cells pellets were incubated for 12 h at − 80 °C. RNAs were extracted using the “Direct-zol RNA miniprep kit” (Zymo Research, Irvine, CA, USA). Genomic contaminations were removed by treatment with Turbo DNase according to the manufacturer recommendations (ThermoFisher). For cDNA synthesis, 1 μg of RNAs was reverse transcribed with the “BIORAD iScript Select cDNA synthesis Kit” kit (Bio-Rad, Hercule, CA, USA). For each condition, RT-qPCR experiments have been carried out using three independent RNA samples. Primers (5′ to 3′) are listed in Table S2. For gene expressions, transcript levels were determined by the DeltaDelta Ct method using the adk gene as a housekeeping control gene. The Student t-test was used to determine the statistical significance between the samples. For all comparisons, fold changes (FC) > 2 or < − 2 with a p-value less than 0.05 were considered as significant.