Ampigene qpcr green mix

The mRNA of the HDFs was extracted by using TRIzol™ (Invitrogen, CA, USA) according to the manufacturer’s protocol. The mRNA sample (1 mg) was reverse transcribed to cDNA by using amfiRivert cDNA Synthesis Platinum master Mix (GenDEPOT, Barker, TX, USA). Relative gene expression was analyzed with the 7500 Real-Time PCR system (Applied Biosystems, Foster City, CA, USA). Reaction mixture with each primer (Bioneer, Daejeon, Korea) and AMPIGENE^® qPCR Green Mix (Enzo Life Sciences, Farmingdale, NY, USA) were applied to 40 cycles of RT-PCR (95 °C for 15 s, 60 °C for 60 s, and 95 °C for 10 min). The relative gene expression against the GAPDH gene was evaluated based on the comparative or ΔΔCt method. Table 1 lists the primer sequences used in this study.

Reverse-transcribed using 5X All‐in‐One RT Master Mix (ABM, No. G492). Quantitative real‐time polymerase chain reaction (qRT‐PCR) was performed using AMPIGENE qPCR Green Mix (Enzo Life Sciences, No. ENZ‐NUC104‐1000). RN18S was used as an endogenous control. The qRT-PCR primer sequences used in this study are listed in Supplementary Table S1.

JBP muscle cells were collected at passages of P6 to P10. Total RNAs were extracted from these cells using an AccuZol Total RNA extraction kit (Bioneer, Daejeon, Korea). Quantity and purity of RNAs were determined using a spectrophotometer (μDrop plate, Thermo Fisher Scientific, USA). One microgram of total RNA was reverse-transcribed to cDNA with a cDNA synthesis kit (Bioneer, Korea). Quantitative real-time polymerase chain reaction (qRT-PCR) assays were performed using 1 μL of cDNA and 19 μL of stock solution containing AMPIGENE qPCR Green Mix (Enzo, San Diego, CA, USA), UltraPure distilled water (Invitrogen, USA), and primer solution containing both sense and antisense custom-designed primers on a CFX96 real-time PCR detection system (Bio-Rad, USA). Samples were denatured at 95°C for 5 minutes and cycled 40 times at 95°C (for denaturing) for 5 seconds and 60°C (for annealing and extension) for 30 seconds. Gene-specific primer sequences are listed in Table 1. The qRT-PCR results were normalized against GAPDH as a housekeeping gene to calculate the expression of each target gene.

The mRNA expression levels of S100A8 (signature gene of psoriasis) and PD-L1 in the lesional and non-lesional skin superficial tissues of 11 patients with CPP were analyzed using qRT-PCR. Total RNA was extracted using the total RNA Mini kit (Favogen, Pingtung, Taiwan). The RNA (1 μg) was reverse transcribed into complementary DNA using a RevertAid first-strand cDNA synthesis kit (Thermo Scientific). The qRT-PCR analysis was performed using LightCycler^® 480II with AMPIGENE qPCR Green Mix (Enzo Life Sciences, Farmingdale, NY). The PCR conditions were as follows: 95 °C for 5 min (initial denaturation), followed by 45 cycles of 95 °C for 10 s, 60 °C for 10 s, and 72 °C for 10 s. The primer sets used in the qRT-PCR analysis are listed in the Supplementary Materials (Table S1).