Topscript cdna synthesis kit

A total of five to ten wing discs were homogenized in 100 μL of PBST (PBS with 0.1% Triton x-100). Total RNA was extracted from imaginal wing discs of the third instar Drosophila larvae using the TRIzol reagent (Invitrogen, ThermoFisher Scientific), followed by a synthesis of cDNA from 2 μg of extracted RNA using TOPscript^TM cDNA synthesis kit (Enzynomics, Inc., Daejeon, Korea), according to the manufacturer’s instructions. A standard RT-PCR analysis was performed using PrimeScript RT Master Mix (Takara Bio Inc.), according to the manufacturer’s instructions. Mir-X^TM miRNA First-Stand Synthesis Kit (Qiagen Inc., Germantown, MD, USA) was used for amplification of the coding region of mature miRNAs. Each RT-PCR reaction was followed by gel electrophoresis and visualization using Multiple Gel DOC system (Fuji Photo Film Co., Ltd., Tokyo, Japan).

Total RNA was isolated using TRIzol reagent (Invitrogen) according to the manufacturer's protocol. mRNA was reverse‐transcribed into complementary DNA (cDNA) using TOPscriptTM cDNA Synthesis kit (Enzynomics, South Korea). Quantitative PCR analysis was performed using the Power SYBR Green PCR Master Mix (Applied Biosystems) with a StepOnePlus Real‐Time PCR System (Applied Biosystems) according to the manufacturer's instructions. Target gene expression was normalized to the glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) gene for quantification. Primer sequences used for qRT‐PCR analysis are shown in Table 1.

Total RNA was isolated from various MSCs cultures using the Direct-zol™ RNA MiniPrep (Zymo Research Corporation, Irvine, CA, U.S.A.) according to the manufacturer’s protocol. RNA concentrations were determined using a NanoDrop spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA), and reverse transcription reactions were performed using 0.2 μg of total RNA with a TOPscriptTM cDNA synthesis kit (enzynomics, Daejeon, Korea). The real-time PCRs for beta-actin, collagen II, and aggrecan were performed using the TOPrealTM qPCR 2X Pre MIX (enzynomics). Primer sequences are listed in Table 1. Real-time PCRs were performed using a StepOnePlus™ instrument (Applied Biosystems, Grand Island, NY, USA) at 95 °C for 15 min followed by 40 cycles of denaturation at 95 °C for 10 s, extension at 60 °C for 15 s, and annealing at 72 °C for 15 s. Gene expression levels were normalized to that of beta-actin and relative gene expression was calculated using the ddCT method.

Total RNA was extracted from HaCaT cells and mouse right ears (n = 7 per group) using TRIzol reagent (Thermo Fisher Scientific, Waltham, MA, USA) and reverse-transcribed with the TOPscript^TM cDNA Synthesis kit (Enzynomics, Seoul, Korea). The synthesized cDNA was mixed with Dyne 2X PreMIX-HOT (DYNE Bio, Seongnam, Korea) and gene-specific primers. The amplification conditions were as follows: initial melt at 95 °C for 3 min, followed by 22 to 35 cycles of denaturation at 95 °C for 30 s, annealing at 58–60 °C for 45 s, extension at 72 °C for 45 s, and final extension at 72 °C for 10 min. The amplified products were evaluated by 1% agarose gel electrophoresis. The density of PCR products obtained from mouse ears was measured using ImageJ software (v 1.50i, National Institutes of Health, Bethesda, MD, USA). The primers used for amplification of candidate genes are presented in Table S2.

B16F10 cells were incubated with a growth medium containing Ech A (0, 0.1, 0.5, 1, 5, and 10 μM) or 50 μM kojic acid at 37 °C and 5% CO₂ for 1 h. Then, 100 nM α-MSH was added, and the cells were incubated at 37 °C and 5% CO₂ for an additional 23 h or 47 h. To analyze the expression of the Tyr, Tyrp1, Tyrp2, and Mitf mRNA, we performed qRT-PCR. The cells exposed to Ech A or kojic acid for 24 h and 48 h were harvested, and total RNA was extracted using a Trizol reagent (Thermo Fisher Scientific, Waltham, MA, USA). The extracted total RNA was reverse-transcribed with a TOPscript^TM cDNA Synthesis Kit (Enzynomics, Seoul, Korea). For quantitative PCR, 5 µL of TB Green Premix Ex Taq (Takara Bio, Otsu, Japan), 5 pmol of the forward primer, 5 pmol of the reverse primer, and 1 µL of cDNA were added with water to a final volume of 25 µL. The mixture was amplified for 35 cycles using a CFX96 Touch Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). The cycle number at which a statistically significant increase in each gene was first detected (threshold cycle, Ct) was normalized to the Ct for Gapdh. The relative expression differences between the genes were calculated using the 2^−ΔΔCT method [44 (link)]. The primers used for gene amplification are presented in Table 1.

First, 1 × 10⁵ of HCT116 cells seeded in a 6-well plate were treated with either DMSO or GE and incubated for 24 h. Total RNA was purified using a GeneJET RNA purification kit (Thermo Scientific). From the purified RNA, cDNA was synthesized using a TOP scriptTM cDNA synthesis kit (Enzynomics, Daejeon, Korea). All of the primers used in this study were obtained from Macrogen (Seoul, Korea) and are listed in Table S2. The detailed protocol for PCR included 28 cycles of denaturation at 94 °C for 45 s, annealing at 57 °C for 45 s, and elongation at 72 °C for 45 s. A volume of 10 μL of the PCR product was electrophoresed on a 1.5% agarose gel and visualized using a MiniBIS Pro gel documentation system (DNR Bioimaging Systems, Neve Yamin, Israel). For RT-qPCR, cDNA was amplified using Power SYBR Green PCR Master Mix (Thermo Scientific) on a QuanStudio 1 real-time PCR instrument (Applied Biosystems, Waltham, MA, USA). Relative mRNA levels were normalized to GAPDH mRNA levels. The relative expression levels were determined by comparative Ct method. Primer sequences are listed in Table S2.

The Gene JET RNA Purification Kit (Thermo Scientific, Waltham, USA) was used to extract total RNA from the adipose tissues according to the manufacturer’s instructions. The isolated RNA was reverse transcribed into cDNA using the TOPscriptTM cDNA Synthesis Kit (enzynomics, Daejeon, Korea). The expression of RBP4 and GLUT4 was measured using a QIAGEN Rotor-Gene Q 5plex system (QIAGEN, Hilden, Germany) with TOPrealTM qPCR 2X PreMIX (SYBR Green with low ROX) (enzynomics, Daejeon, Korea) according to the manufacturer’s guidelines. Primers were purchased from Willowfort (Birmingham, UK), and the sequences are presented in Table S1.
The cycling conditions for real-time PCR included an activation step at 95 °C for 15 min followed by 45 cycles of 95 °C for 10 s, 65 °C for 15 s, and 72 °C for 30 s. A melting curve analysis was conducted, and the expression of RBP4 and GLUT4 was normalized against that of GAPDH. The results are displayed as fold-change compared with the control group. The relative concentration (fold-change) of RBP4 and GLUT4 mRNA was computed using the 2^−∆∆CT method [35 (link)].