Abi 7500 real time fluorescent quantitative pcr system

The qRT-PCR analysis was performed as described previously (21 (link)), with minor modifications. Isolates were incubated without any drug or with PG alone, FLC alone, or PG+FLC at synergistic concentrations at 37°C overnight. The suspensions were adjusted to 5 × 10⁷ cells/ml in PBS, and the supernatants were collected after centrifugation at 3,000 × g. Total RNA was isolated using a yeast RNAiso reagent kit (TaKaRa, Shiga, Japan) according to the manufacturer’s instructions. RT-PCR was performed using RevertAid first-strand cDNA synthesis kits (Thermo Fisher Scientific, Waltham, MA, USA). qRT-PCRs for CgCDR1, CgCDR2, and CgPDR1 were run in triplicate using SYBR green real-time PCR master mix kits (Toyobo, Osaka, Japan) in an ABI 7500 real-time fluorescent quantitative PCR system (Applied Biosystems, Foster City, CA, USA). The primers used in this study are listed in Table 4. Each qRT-PCR mixture (25 μl) contained 12.5 μl SYBR green real-time PCR master mix, 9.5 μl double-distilled water, 2 μl each primer, and 1 μl cDNA. PCR conditions were as follows: initial denaturation at 95°C for 1 min, followed by 40 cycles of 15 s at 95°C, 15 s at 60°C, and 45 s at 72°C. Target gene expression was quantified using the 2^–ΔΔCT method, with ACT1 as a control (39 (link)).

After RAW 264.7 cells were treated with LPS and different concentrations of MLFB for 24 h, the mRNA expressions of IL-1β, IL-6, TNF-α, NF-κB, PGE₂, TLR-4 in RAW 264.7 cells were determined by RT-qPCR (6 (link)). Firstly, TRIzol reagent was used to extract the total RNA of cells. Then RNA was synthesized into cDNA by cDNA reverse transcription kit referring to the manufacturer's descriptions. Finally, the RT-qPCR was proceeded in 20 μL reaction system containing 2 μL cDNA, 1 μL primer pairs (10 μmol/L), 10 μL SYBR Green PCR Master Mix, 0.4 μL 50 × ROX Reference Dye 2 and 6.6 μL ultra-pure distilled water. The PCR conditions were as followings: initial denaturation at 95°C for 5 min, 40 cycles at 95°C for 10 s, 60°C for 34 s, and the melting curve was obtained at 95°C for 15 s, 60°C for 1 min and 95°C for 15 s in the ABI 7500 real-time fluorescent quantitative PCR System (Applied Biosystems, Foster, CA, USA). β-actin was used as internal control and the primers used here are shown in Table 1.