Sv total rna isolation kit

RNA was extracted from three biological replicates of each strain to be tested using a Promega SV total RNA isolation kit according to the manufacturer's instructions (Promega, Southampton, United Kingdom). RNA and contaminating DNA concentrations were determined using a Qubit fluorimeter (Life Technologies, Paisley, United Kingdom). Residual DNA contamination was removed by treatment with Turbo DNase (Life Technologies), and cDNA was generated using SuperScript III reverse transcriptase (Life Technologies) following the first-strand synthesis protocol supplied by the manufacturer. Expression of patA and patB relative to the expression of rpoB was measured by quantitative real-time PCR by monitoring the fluorescence of SYBR green dye. Reaction mixtures consisted of 12.5 μl IQ SYBR green supermix (Bio-Rad, Hemel Hempstead, United Kingdom), 375 nM (each) forward and reverse primers, and 1 μl cDNA in a 25-μl reaction mixture. Real-time PCR was carried out using a Bio-Rad CFX96 thermal cycler with the following protocol: 3 min at 95°C, followed by 40 cycles of 10 s at 95°C and 30 s at 54.5°C. Expression values were calculated from the fluorescence data using the Pfaffl method (20 (link)).

Total RNA was extracted from three biological replicates of logarithmic-phase cultures of each test strain using a Promega SV Total RNA Isolation kit according to the manufacturer's instructions (Promega, Southampton, UK). RNA and contaminating DNA concentrations were measured using a Qubit fluorimeter according to the manufacturer's instructions (Life Technologies, Paisley, UK). Residual DNA contamination was removed by treatment with Turbo DNase (Life Technologies), and cDNA was generated using Superscript III reverse transcriptase (Life Technologies) following the first-strand synthesis protocol supplied by the manufacturer. Expression of patA, patB, spr1886 and guaA was measured relative to expression of rpoB by SYBR Green Quantitative real-time (qRT) PCR. Reactions consisted of 12.5 μL IQ SYBR Green Supermix (Bio-Rad, Hemel Hempstead, UK), 375 nM each of forward and reverse primers and 1 μL cDNA in a 25 µL reaction. Real-time PCR was carried out using a Bio-Rad CFX96 thermal cycler with the following protocol: 3 min at 95°C, followed by 40 cycles of 10 s at 95°C and 30 s at 54.5°C. Expression values were calculated using the Pfaffl method.^{17 (link)}

To elucidate the effect of DMTU on biofilm- and virulence-related genes of P. gingivalis, quantitative Real Time PCR (qRT-PCR) was performed. Multispecies biofilms were developed in the presence of a biofilm inhibitory concentration of DMTU (0.79 µM) as mentioned above. The planktonic cells were removed by washing twice with PBS. The biofilms were then scraped and centrifuged at 14,000× g for 10 min. Total RNA was extracted from the pellet as per the manufacturer’s instructions using the Promega SV total RNA isolation Kit (Promega, Madison, WI, USA). Using Nanodrop, the purity and concentration of RNA were determined. RNA was reverse transcribed to cDNA using High-Capacity cDNA Reverse Transcription kits (Applied Biosystems, Foster City, CA, USA).
The sequence of primers used in this study is listed in Table S1. Each PCR reaction was performed with a total reaction volume of 20 µL containing 10 µL of SYBR green master mix, 1 µL each of forward and reverse primers, 1 µL of diluted cDNA and 4 µL of nuclease-free water. 16S rRNA was used as a house-keeping gene and to calculate the relative changes in gene expression. Gene expression changes were calculated using the 2^−ΔΔCt method and expressed as a reduction in relative fold change compared to the control.