Thermal cycler dice tp850

The qPCR was carried out to measure cytokine expression using the Thermal Cycler Dice TP850 real-time system (Takarabio, Shiga, Japan) according to the manufacturer’s protocol. At the end of the animal experiment, the entire left hind paw was excised after removal of the skin, and total RNA was isolated. Human RA synovial fibroblasts (2 × 10⁵ cells/well in 24-well plates), were pretreated with 4-HMC or Dexa for 1 h, followed by stimulation with TNF-α (10 ng/mL) for 12 h. Total cellular RNA was isolated from the cells using RNAiso Plus (Takarabio), and cDNA was synthesized using RT Premix (iNtRON Biotech, Sungnam, Korea). Reverse transcription was conducted at 45°C (60 min) and 95°C (5 min). Briefly, 2 μL of cDNA (100 ng), 1 μL each of sense and anti-sense primer solutions (0.4 μM), 12.5 μL of SYBR Premix Ex Taq (Takarabio), and 9.5 μL of dH₂O were mixed to obtain the final reaction mixture (25 μL). The list of primers is shown in Supplementary Table S2. mRNA expression was normalized and quantified using the TP850 software supplied by the manufacturer.

Bacteria were cultured in LB with shaking to OD₆₀₀ of 1.2 and 1.8 for mid-exponential and stationary phases, respectively. Total RNA was extracted using a RNeasy Mini Kit (Qiagen, Valencia, CA). cDNA was synthesized by reverse transcription with 1.5 μg of total RNA using random hexamer primer and RevertAid reverse transcriptase in a total reaction volume of 20 μl (Thermo Fisher Scientific, Waltham, MA). Specific primers for the zrlA gene, 5′-CCC AGC CGA CGA TTA CTC AT-3′ and 5′-GCG ATC CAA ACG ACA TAA TCT TC-3′, and 16S rRNA gene, 5′-GCA CAA GCG GTG GAG CAT-3′ and 5′-CGA AGG CAC CAA TCC ATC TC-3′, were used. Gene transcripts was quantified using a Thermal Cycler Dice TP850 (Takara Bio, Shiga, Japan) with SYBR Premix Ex Taq (Takara Bio) following the manufacturer’s instructions. Amplification specificity was evaluated using melting curve analysis. Gene expression was normalized to 16S rRNA expression in each sample, and fold change was analyzed using the ΔΔCt method.

The expression of cytokines was determined by performing qPCR in a Thermal Cycler Dice TP850 (Takara Bio, Shiga, Japan) according to the manufacturer’s protocol. At the end of the in vivo experimental period, the mouse ears (n = 20) were excised, and the total RNA was isolated. HaCaT cells were pretreated with LLK for 1 h, followed by stimulation with DFE (100 μg/mL) for 6 h, 12 h, or 24 h. The total cellular RNA was isolated from cells (2 × 10⁵ cells in 24-well plates) using RNAiso Plus (Takara Bio). The first strand complementary DNA (cDNA) was synthesized using RT Premix (iNtRON Biotech, Sungnam, Korea). The reverse transcription conditions were 45 °C for 60 min and 95 °C for 5 min. qPCR was performed in a 25 μL reaction mixture containing 2 μL cDNA (100 ng), 1 μL sense and antisense primer solution (0.4 μM), 12.5 μL SYBR Premix Ex Taq (Takara Bio), and 8.5 μL distilled water. The conditions for qPCR were similar to those used previously [19 (link)]. Primers are listed in Supplementary Table S2. The quantification and normalization of mRNA levels were performed using TP850 software supplied by the manufacturer. β-actin was used for normalization.

Total RNA from cells was collected using RNeasy Plus Micro Kit (QIAGEN, Venlo, Netherlands) according to the manufacturer's instruction and the concentration of total RNA was measured using a Q5000 (Tomy Digital Biology, Tokyo, Japan). Total RNA (0.5 μg) was used for reverse transcription using SuperScript IV Reverse Transcription (Thermo Fisher Scientific). Quantitative PCR was performed using Thermal Cycler Dice TP850 (Takara Bio). The PCR primers are #12 for mCherry and #13 for human GAPDH. One of the 0 h cDNA samples was step-diluted by 0.5-fold and used as a standard for quantification. The relative value of mCherry divided by the relative value of GAPDH was averaged for the DMSO- and ASV-supplemented groups.

Prior to isolation of total cellular RNA, HMC-1 cells (5 × 10⁵/well in 24-well plates) were pretreated with or without compound for 1 h and then stimulated with PMA (40 nM) and CI (1 μM) for 1 h. RNAiso Plus reagent (Takara Bio Inc., Shiga, Japan) was used to extract total RNA, in accordance with the manufacturer’s protocol. Complementary DNA (cDNA) was synthesized from 2 μg of total RNA using the Maxime RT-Pre Mix Kit (iNtRON Biotechnology, Daejeon, Korea). Quantitative real-time PCR was carried out using the Thermal Cycler Dice TP850 (Takara Bio Inc.) according to the manufacturer’s protocol. The 25 μL reaction mixture was composed as follows: 1.5 μL of cDNA (150 ng), 1 μL of each of the forward and reverse primers (0.4 μM), 12.5 μL of SYBR Premix Ex Taq (Takara Bio Inc.), and 9 μL of D₂O [15 (link)].

After stimulation with DNA-HSA with or without AEDKC, cells were seeded at a density of 5 × 10⁵ cells/well in a 12-well plate. Total cellular RNA was isolated using a RNAiso Plus kit (Takara Bio, Shiga, Japan) according to the manufacturer’s protocol. The first strand complementary DNA (cDNA) was synthesized using a PCR kit (Thermo Scientific). The primer sets were chosen by the Primer3 program (Whitehead Institute, Cambridge, MA). The cycle number was optimized to ensure product accumulation was in the exponential range. qPCR was carried out using a Thermal Cycler Dice TP850 (Takara Bio) according to the manufacturer’s protocol. Briefly, 2 μL of cDNA (100 ng), 1 μL of sense and antisense primer solution (0.4 μM), 12.5 μL of SYBR Premix Ex Taq (Takara Bio), and 9.5 μL of nuclease free water were mixed together to obtain a final volume of 25 μL in each reaction tube. Relative quantification of mRNA expression was performed using the TP850 software. The conditions used for PCR were as previously described (Bae et al. 2011 (link)).