Topreal qpcr 2 premix kit

Cells (1 × 10⁵ cells/mL) were seeded in cell culture plates and then treated with ampelopsin (0, 50, and 100 µM) for 24 h. TRIzol reagent (Invitrogen, Carlsbad, CA, USA) was then used to extract total RNA. After reverse transcription of extracted total RNA, TOPreal™ qPCR 2× PreMIX kit (Enzynomics, Daejeon, South Korea) was used to conduct real-time PCR. The primers used are listed in Table S1. The 2^−ΔΔCt method described by Livak and Schmittgen was used to evaluate the gene expression [61 (link)].

Total RNA was extracted from cancer cells using TRIzol (Invitrogen) reagent and reversed-transcribed into cDNA using ImProm-II^TM Reverse Transcriptase (cat. no. A3802; Promega, Madison, WI, USA) following the manufacturer’s instructions. Then, two-step quantitative real-time PCR was performed (Thermal Cycler Dice Real-Time System; Takara, Shiga, Japan) using a TOPreal™ qPCR 2× PreMIX kit (Enzynomics, Daejeon, Korea). The cycling conditions were as follows: initial hold at 95 °C for 15 min, and 40 cycles at 95 °C for 10 s and 60 °C for 30 s. A dissociation curve was generated (95 °C for 15 s, 60 °C for 30 s, and 95 °C for 15 s). The real-time PCR primers are listed in Table S1. GAPDH was used as an endogenous control. Expression levels were calculated using the 2^−ΔΔCq method. Data were represented after three-time biological and three-time technical replicates [23 (link)].

Total RNA was extracted from cells that were pretreated with SA (100, 200, and 400 μM) followed by treatment with 50 μM of 6-OHDA using the Ribospin™ II kit (GeneAll Biotechnology Co., LTD, Seoul, Korea). After measuring the total RNA concentration using a UV/Vis Nano Spectrophotometer (MicroDigital Co., Ltd., Sungnam, Korea), 1 μg of total RNA from each group was reverse transcribed to synthesize cDNA using the GoScript Reverse Transcription System Kit (Promega Co., Madison, WI, USA). qRT-PCR was performed using a TaKara Thermal Cycler Dice Real-Time System (Takara Bio. Inc., Shiga, Japan) using the TOPreal™ qPCR 2× PreMIX Kit (Enzynomics Co., Daejeon, Korea). The primer sequences are as shown in Table 1.