RNA Preparation and qRT-PCR Experiments

RNA was isolated from mycelia obtained from S. lividans TK24 (SL), wild type, its phoP mutant and S. coelicolor M145 (SC) grown for 40 h at 28°C on the solid R2YE medium with no K₂HPO₄ added (containing 1 mM free phosphate from elements of the media as determined with PiBlue^TM Phosphate Assay Kit from BioAssays Systems). In order to preserve RNA integrity, the mycelium was immediately freezed in liquid nitrogen in a solution containing denaturating guanidinium thiocyanate buffer RA1 (Macherey-Nagel, Hoerdt, France), phenol-chloroform and ß-mercaptoethanol (a reducing agent). The cells were then lysed and homogenized in the presence of glass beads (diameter < 106 μm) using a Fast-Prep apparatus (Savant Instruments). Total RNA was purified using the Nucleospin RNA Kit (Macherey-Nagel, Hoerdt, France), according to the manufacturer’s instructions. To remove residual DNA, a DNAse TURBO^TM treatment (Invitrogen) was performed at 37°C for one hour and total RNA was purified with the Nucleospin RNA Clean-Up kit (Macherey-Nagel, Hoerdt, France). The RNA concentrations were quantified using the Nanodrop 2000 spectrophotometer (Thermo Scientific). The integrity of the RNAs was verified using the Agilent 2100 bioanalyzer with the eukaryote total RNA 6000 Nano assay (Agilent Technologies). A total of 1 μg of total RNA was reverse transcribed in a 20 μL final reaction volume using the High Capacity cDNA Reverse Transcription Kit (Life Technologies) with RNase inhibitor and random primers following the manufacturer’s instructions. Quantitative PCR was performed on a QuantStudio 12K Flex Real-Time PCR System (Life Technologies) with a SYBR green detection protocol. A total of 3 ng of cDNA were mixed with Fast SYBR Green Master Mix and 750 nM of each primer in a final volume of 10 μL. The reaction mixture was loaded on 384 well microplates and submitted to 40 cycles of PCR (95°C/20 sec; [95°C/1 s; 60°C/20 s] X40) followed by a fusion cycle to analyze the melting curve of the PCR products. A qPCR analysis in the absence of a reverse transcription step was performed on all RNA samples to check the absence of any DNA contamination. Primers were designed using the Primer-Blast tool from NCBI and the Primer Express 3.0 software (Life Technologies) (Supplementary Table S1). Specificity and the absence of multi-locus matching at the primer site were verified by BLAST analysis. The amplification efficiencies of primers were generated using the slopes of standard curves obtained by a 10-fold dilution series. Amplification specificity for each real-time PCR reaction was confirmed by analysis of the dissociation curves. Each sample measurement was made in duplicate and four independent RNA biological samples were prepared for each condition. Determined cycle threshold (Ct) values were then exploited for further analysis. Cycle threshold is defined as the calculated cycle number at which the PCR product crosses the threshold of detection. This value tells how many cycles it took to detect the signal from your samples. Seven most stable reference genes were selected by GenEx software (MultiD) and the geometric mean of the five most stable genes (Glk/SCO2126, AspS/SCO3795, GyrA/SCO3873, GyrB/SCO3874, and RpoB/SCO4654) was used to normalize the data (HrdB/SCO5820 and RecG/SCO5566 were excluded). The determination of the relative gene expression ratio was achieved using the ΔΔCt method using three biological replicates (Pfaffl, 2001). The values of ΔΔCt of SC and of the phoP mutant of SL were normalized and standardized by log transforming, mean centering and autoscaling (Willems et al., 2008). All data were subjected to the Student test and the results were presented as the mean of delta-delta-Ct with error bars representing 95% confidence interval.