Lightcycler 480 sybr green 1 master

THP-1 cells (2 × 10⁵) were treated with either iDual, iHDAC6, or GSK2879552 (5 μM in each case) or ricolinostat (1.25 μM) for 72 h and compared to vehicle control (DMSO). NucleoSpin™ RNA Plus Isolation Kit (Macherey Nagel™, Dueren, Germany, 740955.50) and M-MLV Reverse Transcriptase kit (Thermo Fisher Scientific, Waltham, MA, USA, #28025013) protocols were employed for RNA isolation and cDNA synthesis, respectively, with 500 ng of RNA converted to cDNA for each sample. Gene expression levels were quantified with qRT–PCR method using a LightCycler 480 SYBR Green I Master (Roche Diagnostics, Penzberg, Germany, #50-720-3180) and normalized to β-actin or GAPDH. Briefly, 10 ng of cDNA template was amplified using 10 μL LightCycler 480 SYBR Green I Master (Roche Diagnostics), and gene-specific primers (Table S1) mixed in a final volume of 20 μL. The samples were incubated at 95 °C for 5 minutes, followed by 40 cycles of 95 °C for 10 seconds, 55 °C for 30 seconds, and 72 °C for 1 second. The relative amounts of gene expressions were calculated with the 2^(−ΔΔCT) method. All analyses were performed in duplicates and three biological replicas.

Total RNA of T. chilonis was extracted using the TRIzol method (Taraka, Japan). To obtain the first-strand cDNAs, 1 μg of total RNA was used for reverse transcription in a reaction system with a total volume of 20 μL, according to the manufacturer's instructions (PrimeScript RT Reagent Kit, TaKaRa, Japan). qRT-PCR was performed using LightCycler480 SYBR-Green I Master (Roche Diagnostics, Basel, Switzerland) and run on the LightCycler480 Real-time PCR system (Roche Diagnostics Ltd). Each reaction was conducted in a reaction system with a total volume of 10 μL with 1 μL of cDNA (2 ng/μL), 5 μL of SYBR Green I Master (LightCycler480 SYBR Green I Master, Roche Diagnostics Ltd., Lewes, UK), 0.5 μL/primer, and 3 μL of ddH₂O. The qRT-PCR was conducted using the following programme: denaturation at 95°C for 5 min, followed by 40 cycles of 5 s at 95°C, 20 s at 60°C, and 20 s at 72°C. gapdh was the internal reference gene. Each gene was tested in triplicate, and the experiments were conducted on three biological replicates. The relative expression levels of the genes normalized to the internal control gene, were calculated using the 2^-ΔΔCt method [54 (link)]. Analysis of relative gene expression data used a real time quantitative PCR and the 2^-ΔΔCt method.

qPCR was performed in a LightCycler® 480 (Roche) using the LightCycler® 480 SYBR Green I Master (Roche) in 96 wells white PCR microplate and a sealing film (Fisher scientific). The reaction mixture, which was 20 µL in total volume, contained 10 µL of LightCycler® 480 SYBR Green I Master 2 × (Roche), an equal quantity depending on the reaction of forward and reverse primer at 10 µmol/L and 1 µL of cDNA at the appropriate dilution. The mixture was filled to 20 µL by PCR grade water. The PCR program was started from one step of pre-incubation at 95 °C for 5 min, followed by 45 amplification cycles. Each cycle was started by a denaturation step at 95 °C for 10 s and followed by an annealing step at a specific temperature for a specific time depending on the reaction. The amplification step lasted 5 s at 72 °C. The last step was a melting curve analysis. For each run, 2 No Template Controls (NTC) have been performed. The quantity of primers and cDNA as well as the annealing time and temperature were determined to optimize the PCR efficiency (Table S6) as explained below.