Total RNA (RNAs) was extracted from cultured cells using Trizol reagent (Invitrogen, Carlsbad, CA). After that, 2 μg of RNAs was reverse-transcribed into cDNA using SuperScript II Reverse Transcriptase (Invitrogen). One μl of cDNA was diluted into 20 μl of qPCR reaction mixture containing SYBR Green (2xGreen star qPCR MasterMix, Bioneer, Korea). qPCR was performed on Rotor-Gene Q (Qiagen, Hilden, Germany) with cycling conditions as follow: initial denaturation at 95℃ for 10 min, and then 40 cycles of denaturation at 95℃ for 20 s, annealing at 60℃ for 20 s and extension at 72℃ for 25 s. Relative mRNA expression levels of the selected genes were analyzed using ΔΔCT method and were normalized to GAPDH. The sequences of qPCR primers are listed in Table 1 .
2xgreen star qpcr mastermix
Manufactured by Bioneer
2xGreen star qPCR MasterMix is a ready-to-use solution for real-time quantitative PCR (qPCR) reactions. It contains all the necessary components, including a proprietary green fluorescent dye, required for the amplification and detection of DNA targets.
Automatically generated - may contain errors
Lab products found in correlation
Cyclescript rt pre master mix, by Bioneer (2 mentions)
Accuprep universal rna extraction kit, by Bioneer (2 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Superscript 2 reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Rotor gene q, by Qiagen (1 mentions)
4 protocols using 2xgreen star qpcr mastermix
1
Quantitative RT-PCR Protocol for Gene Expression
2
Quantifying mRNA Expression in Cartilage and Cell Cultures
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Total RNA was extracted from OA-induced cartilage tissues (articular cartilage and meniscus) and LPS-stimulated RAW264.7 cells using the AccuPrep® universal RNA Extraction Kit (Bioneer corp., Korea) and then reverse-transcribed into cDNA using the CycleScript™ RT Pre&Master Mix (Bioneer, Korea), following the manufacturer’s protocol. mRNA expression was quantified using 2X-GreenStar qPCR MasterMix (Bioneer, Korea) (Lu et al., 2018 (link); Lee et al., 2020 (link)). This experiment was repeated triplicated. The primer sequences are listed in Supplementary Tables S1, S2 .
3
Quantitative Gene Expression Analysis
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Total RNA was extracted from OA-induced cartilage tissue and LPS-stimulated RAW264.7 cells using an AccuPrep® Universal RNA Extraction Kit (Bioneer, Daejeon, Republic of Korea) and reverse transcribed into cDNA using CycleScript™ RT Pre&Master Mix (Bioneer, Republic of Korea), according to the manufacturer’s protocol. mRNA expression was quantified with 2X-GreenStar™ qPCR MasterMix (Bioneer, Republic of Korea). All experiments were repeated thrice. The relative gene expression was determined using the threshold cycle (CT) method, and the fold changes were calculated using the 2−ΔΔCT formula. The primer sequences consisted of exons and are listed in the following Table 6 and Table 7 :
4
Cell Culture Optimization and Gene Expression Analysis
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B16-F10 and HaCaT cells (5 × 104 cells/well) were plated on 12-well plates and incubated. Then, B16-F10 cells were treated with 1% (v/v) CFS or arbutin (200 µM) for 6 h and 40 h in the presence or absence of 200 nM α-MSH. HaCaT cells were pretreated with CFS (1 and 3% (v/v)) for 30 min and then co-treated 100 µM H2O2 for 24 h. Then, the cells were harvested and washed twice with phosphate-buffered saline. Total cellular RNA was prepared using TRIzol solution according to the manufacturer’s instructions. RNA was converted to cDNA using RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific), according to the manufacturer’s instructions. The 2X GreenStar qPCR Master Mix (Bioneer, Daejeon, Korea) was used in all the samples, and reactions were carried out in a 20 µL final reaction volume. Each experiment was performed at least twice in duplicates using the following primers in Supplementary Table S1 . All gene expression levels were calculated by using the Ct value by the method 2−ΔΔCt (where ΔCt = Ct[target gene] − Ct[GAPDH]).
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