Tb green premix ex taq 2

The transcript levels of osteoclast-related genes were detected by RT-qPCR. BMMs (2 × 10⁵ cells/well) were seeded into 6-well plates supplemented with M-CSF (30 ng/mL), RANKL (100 ng/mL), and different concentrations of ArA (0, 10, 20, 40, and 80 μM) for 3 days. The TRIzol reagent was used to lyse the cells and extract total RNA. The entire experimental process of RT-qPCR strictly followed the MIQE guidelines (Taylor et al., 2010 (link)). RNA concentration and purity were determined using a Nanodrop 2000 instrument (Thermo Fisher Scientific, Waltham, MA, United States), ensuring that OD260/280 was between 1.8 and 2.0. Equal amounts of RNA (1,000 ng) were then reverse transcribed to synthesize complementary DNA (cDNA) to avoid inflating the differences between groups. According to the manufacturer instructions for TB Green™ Premix Ex Taq™ II, a 10 μL reaction system was established and used for the RT-qPCR assay on an ABI 7500 Sequencing Detection System (Thermo Fisher Scientific). Cycling conditions were set to 40 cycles (95°C for 5 s and 60°C for 30 s). Melting curves were examined to verify amplification specificity. Relative gene expression was calculated using the comparative 2^−ΔΔCT method (Livak and Schmittgen, 2001 (link)). The primer sequences were as shown in Table 1, among which Gapdh was used as the housekeeping gene.

For real-time RT-PCR, we used a TB Green Premix Ex Taq II (Takara Bio, Shiga, Japan). For each 2 μL of the cDNA sample, 1.6 μL of primer (Forward and Reverse, 0.8 μL each) (Table 1) (Takara Bio) was added, and 8.5 μL of RNase free water, 12.5 μL of TB Green Premix Ex Taq II and 0.4 μL of Rox Reference Dye was added to make a total of 25 μL. The expression level of cDNA was measured using an Applied Biosystems^® 7500 real-time PCR system (Thermo Fisher Scientific). The holding stage was at 50 °C for 2 min and 95 °C for 10 min. The cycling stage for 40 cycles was at 95 °C for 15 s, and 60 °C for 1 min. The melt curve stage was at 95 °C for 15 s, 60 °C for 1 min, 95 °C for 30 s, and 60 °C for 15 s. The analysis of the gene expression level of each sample was carried out in comparison with the expression level of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), which is a housekeeping gene. The expression levels of DUSP1 were calculated as correction values divided by the expression level of GAPDH. The primer sequences of each gene used in this study were the same as in the previous report [7 (link)].

RNA was extracted from intestine and liver tissues using the TRIzol reagent as previously described [36 (link)]. We synthesized cDNA from 500 ng of total RNA by using a PrimeScript™ RT Master Mix kit. The reaction was performed in a volume of 20 μL, containing 0.5 ng/μL of cDNA, 400 nM corresponding primer, and 10 μL of TB Green Premix Ex Taq II on an Applied Biosystems QuantStudio 5 Real-Time PCR system (Applied Biosystems, Foster City, CA, USA). The quantitative PCR conditions were 95 °C for 30 s, 95 °C for 3 s, and 60 °C for 30 s for 40 cycles. The sequences of primers were as follows: Mdr1a (forward) 5′-CAACCAGCATTCTCCATAATA-5′, (reverse) 5′-CCCAAGGATCAGGAACAATA-3′; Mdr1b (forward) 5′-CCTCCTTGGTCCTCTCAA-3′, (reverse) 5′-TGTTTGGGGCTAAATGTC-3′; Mrp2 (forward) 5′-GCACATGGCTCCTGGTTTTG-3′, (reverse) 5′-ATACGCCGCATAAGACCGAG-3′; Mrp3 (forward) 5′-TGTGGGTCTTTCCGTGTC-3′, (reverse) 5′-GCCTCAGTCTCCGTCTTAG-3′; and β-actin (forward) 5′-GCCACCAGTTCGCCAT-3′, (reverse) 5′-CATACCCACCATCACACC-3′. The PCR products were analyzed using ΔΔCt with β-actin as the internal standard.

Total RNA extraction, RNA reverse transcription, and real-time fluorescence quantification PCR were carried out with TaKaRa (Takara Biomedical Technology Co., Ltd., Beijing, China) regents according to the manufacturer's instructions. In brief, total RNA was isolated from jejunum and cecal samples using TRIZOL reagent and then treated with DNase I to remove trace DNA. RNA was reverse transcribed to cDNA using the High-Capacity cDNA Reverse Transcription Kit. The real-time quantitative PCR reaction was performed on QuantStudio 7 Flex Real-Time PCR System (Applied Biosystems, Foster City, CA, USA), using TB Green Premix Ex Taq II (Tli RNaseH Plus) with a 10 µL system including 5 µL TB Green Premix, 0.8 µL of primer (forward and reverse primers were premixed), 0.2 µL of ROX Reference Dye II (50 ×), 1 µL of cDNA template, and 3 µL of DNase Free dH₂O [8 (link), 26 (link)]. The primers were commercially synthesized (Invitrogen, Shanghai, China) and are presented in Table S1. Relative gene expression was analyzed by 2^−ΔΔCt method with normalization against the β-actin.

RNA extraction and reverse transcription were performed as per the manufacturer’s instructions (Takara; No. 9109). Then, the resulting cDNA was subjected to real-time qPCR with gene-specific primers (primer sequences are listed in Supplementary Table S4). in the presence of TB Green^® Premix Ex Taq™ II using the StepOnePlus Real-Time PCR System (Applied Biosystems, Thermo Scientific, Waltham, MA, USA). Relative mRNA expression was determined using the 2^−ΔΔCT method with GAPDH or U6 serving as endogenous controls from the mouse samples.