Revertra ace qpcr rt master mix with gdna remover

Total RNA of cultured cells or tissue was extracted with RNAfast200 purification kit (Fastagen), and reverse-transcribed with the ReverTra Ace® qPCR RT Master Mix with gDNA Remover (TOYOBO, FSQ-301). Real-time PCR was performed on the ABI Q3 machine (Thermo) with 2×RealStar Green Power Mixture (GenStar). GAPDH was used as the internal control [31 , 33 (link)]. The sequences of qRT-PCR primers for the genes examined are listed in the supplementary information (Supplementary Table 1).

The mycelia were cultured in GMM, YGMM, or PDB at 37°C and harvested at time-points (18~24 h) appropriate for the applications. The mycelia were frozen in liquid nitrogen, and total RNA was isolated using the FastRNA Pro Red Kit (MP Biomedicals, Santa Ana, CA, USA). To obtain cDNA pools from the total RNA, reverse transcription was performed using the ReverTra Ace qPCR RT Master Mix with gDNA remover (Toyobo, Osaka, Japan).

Total RNA was isolated from mouse primary macrophages and aortas using RNAiso Plus (TaKaRa, D9108A) according to the manufacturer's protocol. RNA quantity and purity were assessed using Nanodrop 1000 spectrophotometer (Thermo Scientific), confirming 260/280 ratio of 1.8 to 2.1 for all samples. 1 μg of total RNA was used for reverse transcription using the ReverTra Ace^® qPCR RT Master Mix with gDNA Remover (TOYOBO, FSQ‐301). The primers (Table S1) were used. Quantitative real‐time PCR was performed using Applied Biosystems^™ SYBR^™ Green (Thermo Scientific^™) on a Real‐Time Detection System (BioRad). The mRNA level was normalized to β‐actin.

All synthetic siRNAs were purchased from Shanghai GenePharma Co. Ltd. The sequences of the siRNAs used are listed in Supplementary Table 1. All siRNAs were transfected with lipofectamine™ 2000 (Invitrogen, 11668-019) according to the manufacturer’s protocol. Total RNA was isolated using RNAiso Plus (Takara, D9108B) according to the manufacturer’s protocol. Real-time qRT-PCR was performed using ReverTra Ace® qPCR RT Master Mix with gDNA remover (TOYOBO, FSQ-301) and SYBR Green PCR Master Mix (TOYOBO, QPK-201). All mRNA levels were measured and normalized to beta-actin. The primers for RT-PCR analysis are listed in Supplementary Table 1.

RAW 264.7 cells were seeded in a 12-well plate (3 × 10⁵ cells/each well) and incubated at 37 °C with 5% CO₂ for 24 h. and stimulated with LPS (1 μg/mL) for 18 h after a 6-h pre-treatment with 5-HMF at different concentrations (0, 31.5, 63.0 and 126.0 μg/mL). Total cellular RNA was isolated from cultured RAW 264.7 cells using Trizol reagent (Sangon Biotech, Shanghai, China). The RNA was reverse-transcribed into cDNA using ReverTra Ace^® qPCR RT Master Mix with gDNA Remover (TOYOBO LIFE SCIENCE, Shanghai, China) in T100™ Thermal Cycler (BIO RAD, Hercules, CA, USA). The sample mixture was prepared with 1.5 μL of cDNA in 10 μL of ChamQ SYBR qPCR Master Mix (Vazyme Biotech, Nanjing, China), primers and ddH₂O for a final volume of 20 μL. The primers were supplied from Sangon Biotech (Shanghai, China) and the sequences are listed in Table 1. The qPCR was carried out using the LightCycler^® 96 Real-Time PCR System (Roche, Switzerland) under the standard thermal cycle conditions: 95 °C for 30 s, 40 cycles of 95 °C for 10 s and 60 °C for 30 s, followed by 95 °C for 10 s, 65 °C for 60 s and 97 °C for 1 s. The threshold cycle (CT) was calculated as the fractional cycle number at which the amount of the amplified target gene reached a fixed threshold. β-actin was used as the housekeeping gene. Each reaction in at least three independent experiments was performed in triplicate.