The samples were centrifuged at 12,000 × g for 15 min at 4°C. The upper layer was transferred to a new Eppendorf tube and 500 μL of isopropanol was added. The samples were mixed https://www.selleckchem.com/products/LDE225(NVP-LDE225).html gently, incubated at room temperature for 15 min and centrifued at 12,000 × g for 10 min at 4°C. The supernatant was removed and the pellet was washed with 75% ethanol. The tubes were centrifuged at 12,000 × g for 10 min at 4°C and the resulting RNA pellets were dried and resuspended in 30 μL of RNase-free water (CP-690550 mouse Fermentas, Villebon sur Yvette, France). These RNA samples were then purified with the RNeasy

MiniKit (Qiagen, Courtaboeuf, France) and checked for yield and quality by measuring the OD ratio at 260, 280 and 320 nm in a BioPhotometer (Eppendorf, Le Pecq, France). Aliquots of 2 μg of RNA were treated with 1 U of DNase I (Fermentas, Villebon sur Yvette, France) to eliminate residual DNA and used for PCR. A control PCR with irrelevant primers BSF8 and BSR1541 was carried out with the RNA to check the absence of any amplification. Total cDNA was then RG7112 supplier synthesized with iScript cDNA Synthesis Kit (BioRad, Marnes la Coquette, France) following the manufacturer’s recommendations. RT-PCR experiments were performed on cDNA with primers tdcf [55] and tyrPLpR (Table

2), and High Fidelity Taq polymerase (Roche, Meylan, France). Quantification of gene expression by real time quantitative PCR Reverse transcription-quantitative real-time PCR (RT q-PCR), with iQ SYBR green supermix (BioRad, Marnes la Coquette, France) and the BioRad CFX96 Real-Time System was used for gene expression analysis. First, primer specificity and efficacy were checked by using 10-fold serial dilutions of L. plantarum IR BL0076 DNA. The melting curves obtained showed the absence of primer dimers, and the calibration curves, for each pair of primers, showed a slope between 86.8% and 96.2%, and a regression coefficient between 0.997 and 1. Total cDNA was serially diluted

(1 in 4), and a 5 μL aliquot was added to each well containing 20 μL of a mix of 12.5 μL of SYBR green supermix, 1 μL of each primer at 7 pmol. μL-1 and 5.5 μL of RNase-free water. The specific primers used to amplify particular cDNA sequences are given in Table 2. ldhD and gyrA are housekeeping genes used to normalize RNA expression data. These genes were used by Duary Mannose-binding protein-associated serine protease et al. [56] as the most stably expressed genes for RT-qPCR experiments in L. plantarum. Moreover ldhD gene was validated in L. plantarum for RT-qPCR experiments by Fiocco et al. [57]. Each run included a negative control with 5 μL of RNase-free water instead of cDNA, and a positive control using L. plantarum IR BL0076 DNA. The amplification program was as follows: 98°C, 30 s and 40 cycles of 95°C, 10 s; 60°C, 30 s. For each experiment, the condition “culture medium 1 (with free tyrosine), OD600nm = 1.0” was used to calibrate the expression data.