Power sybt green pcr master mix

To evaluate the expression of a specific gene, RNA was extracted from adult worms in each group. After collecting the worm pellet, it was ground to a fine frozen powder using liquid nitrogen. After adding a RLT butter, ethanol, and RW1 buffer sequentially to the frozen powdered worm, purified RNA was extracted using RNeasy mini spin column. Finally, RNase-free water was added to the extracted RNA, which was frozen at -80°C until use. Using a NanoDrop (ND-2000, Thermo Fisher Scientific, MA, USA) and an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, USA), the ratios of absorbance at 260-280 nm (OD 260/280) and at 260-230 nm (OD 260/230) were determined and the quality-controlled RNA (OD 260/280 >1.5 and OD 260/230 >1.0) was used for real-time polymerase chain reaction. After first-strand cDNA synthesis using Maxima H minus First strand cDNA Synthesis Kit (Thermo Fisher Scientific, MA, USA), real-time PCR was performed using the cDNA, each gene-specific primer (Table 1), and Power SYBT Green PCR Master mix (Applied Biosystems, MA, USA). Pan-actin was used as a reference gene and the ΔCT was calculated.

B16F10 melanocytes were treated with test compounds for 48 h. Total RNA was extracted using the TRI-Solution™ according to the manufacturer's instructions and quantified using a NanoDrop 2000 spectrophotometer (NanoDrop Technologies). cDNA synthesis was carried out from 1 μg RNA using AccuPower® PCR PreMix (Bioneer) following the manufacturer's recommendation. mRNA expression of the MITF gene, tyrosinase gene, and TRP-1 was quantified using a Power SYBT® Green PCR Master Mix (Applied Biosystems). mRNA levels were normalized with β-actin and fold change of expression was calculated with the ΔΔCT method. The primer sequences were as follows: mouse tyrosinase forward 5′-TACTTGGAACAAGCCAGTCGTATC-3′, reverse 5′-ATAGCCTACTGCTAAGCCCAGAGA-3′; mouse TRP-1 forward 5′-AAACCCATTTGTCTCCCAATGA-3′, reverse 5′-CGTTTTCCAACGGGAAGGTA-3′; mouse MITF forward 5′-GGACTTTCCCTTATCCCATCCA-3′, reverse 5′-GCCGAGGTTGTTGGTAAAGGT-3′. The PCR conditions were 95°C for 2 min followed by 40 cycles of 95°C for 30 s, 60°C for 1 min, and 72°C for 1 min followed by a final 30 sec extension at 72°C. Data were analyzed using the Stepone™ software v2.3 (Applied Biosystems).

B16-F10 melanocytes were treated with test samples for 48 hr. Total RNA was extracted using the TRI-Solution™ according to the manufacturer's instructions and quantified using a NanoDrop 2000 spectrophotometer (NanoDrop Technologies). cDNA synthesis was carried out from 1 µg RNA using AccuPower® PCR PreMix (Bioneer) following the manufacturer's recommendation. mRNA expressions of the MITF gene, tyrosinase gene, and TRP-1 were quantified using a Power SYBT® Green PCR Master Mix (Applied Biosystems). mRNA levels were normalized with β-actin and fold change of expression was calculated with the ΔΔCT method. The primer sequences were as follows: mouse tyrosinase forward 5′-TACTTGGAACAAGCCAGTCGTATC-3′, reverse 5′-ATAGCCTACTGCTAAGCC CAGAGA-3′; mouse TRP-1 forward 5′- AAACCCATTTGTCTCCCAA -TGA-3′, reverse 5′-CGTTTTCCAACGG -GAAGGT A-3′, mouse MITF forward 5′-GGACTTTCCCTTATCCCATCCA-3′, reverse 5′-GCCGAGGTTGTTGGTAAAG -GT-3′. The PCR conditions were 95°C for 2 min followed by 40 cycles of 95°C for 30 s, 60°C for 1 min, and 72°C for 1 min followed by a final 30 sec extension at 72°C. Data were analyzed using the Stepone™ software v2.3 (Applied Biosystems).