Chamq sybr color qpcr master mix

Total RNA was isolated from chondrocytes using TRIzol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. Complementary DNA (cDNA) was synthesized by Hiscript II QRT SuperMix for qPCR (+gDNA wiper) (Vazyme, Nanjing, China) with total RNA. Quantitative PCR was performed using ChamQTM SYBR Color qPCR Master Mix (Vazyme, Nanjing, China) with gene-specific primers. The primer sequences are shown in Supplementary Table 1. The β-actin gene was used as an internal control to normalize differences in each sample.

The HiScript^® II Q RT SuperMix for qPCR (+g DNA wiper) Kit (Nanjing, China) was used according to the manufacturer’s instructions to generate the first-strand cDNA after extracting total RNA from six samples subjected to RNA-Seq. Ten genes were selected to validate the reliability of the RNA-Seq data. The gene-specific primers were produced by Primer 5.0 (Additional file 2: Dataset 1) and synthesized by Sangon Biotech (Shanghai) Co., Ltd. (Shanghai, China). ChamQTM SYBR^® Color qPCR Master Mix (10 μL; Vazyme, Nanjing, China) was mixed with the gene-specific primers, sterilized water and the synthesized cDNA in a total reaction volume of 20 μL. Reactions were performed on a qTOWER 2.2 (Analytik Jena AG, Jena, Germany). The two-step quantitative RT-PCR program was performed at 95 °C for 30 s, followed by 40 cycles of 95 °C for 10 s and 60 °C for 30 s. The 2^−∆∆Ct method was used to calculate the relative expression level of each gene, and actin was selected as the reference gene for normalization (Livak and Schmittgen 2001 (link)). Each reaction was carried out with three biological replicates and three technical replicates.

Total RNA was extracted from cardiac tissue using an RNA isolation kit (RE-03011, FORGENE, China) according to the manufacturer’s instructions. RNA was converted to cDNA using the HiScript III RT SuperMix (R323-01, Vazyme, China). The DNA template was then amplified using a Bio-Rad CFX96TM PCR system and a ChamQ^TM SYBR Color qPCR Master Mix (Q411-02/03, Vazyme, China). Relative gene expression was normalized to RPS11 using the 2^−△△CT method. The detailed sequences of the primers used in this study are listed in (Table 1).Table 1

The primer sequences.

Gene	Forward primer	Reverse primer
Gnas	5′-GGTCTATCCGAGTGTACCCGA-3′	5′-GGCCTTCTCACTATCTCCGTTAAA-3′
Myh7	5′-GACTTCCGGCAGAGGTATCG-3′	5′-AGCCTCTCGGTCATCTCCTT-3′
Nppa	5′-CGGAGCCTACGAAGATCCAG-3′	5′-AAGCTGTTGCAGCCTAGTCC-3′
Nppb	5′-ATCTCAAGCTGCTTTGGGCA-3′	5′-ACAACAACTTCAGTGCGTTACAG-3′
ACTA1	5′-GAGCGTGGCTATTCCTTCGT-3′	5′-GAAACGCTCATTGCCGATGG-3′
PLN	5′-TACCTCACTCGCTCGGCTAT-3′	5′-ATGCAGATCAGCAGCAGACA-3′
CREB1	5′-AGGGCCTGCAGACATTAACC-3′	5′-AGCACTGCCACTCTGTTCTC-3′
Bmp10	5′-ATGGCTGAACTGCGGTTGTA-3′	5′-TTTTACGGTCCACGCCATCA-3′
RPS11	5′-AGATGAAGATGCAGAGGACCATT-3′	5′-GACGCTTCTCAAAGCGATTGT-3′

We performed RT-PCR to confirm the presence of selected DEGs in tissue samples using the primers listed in Supplementary Table 4. RNA samples were reverse transcribed into cDNA using the PrimeScriptTM RT Reagent Kit (Takara, Dalian, China), which was in turn amplified using the ChamQTM SYBR Color qPCR Master Mix (Vazyme, Nanjing, China), and the expression levels were normalized to GAPDH levels. RT-PCR was performed in triplicate for different tissue samples.

The RNA-Quick Purification Kit (Yishan Biotech, Shanghai, China) was used to isolate total RNA from differentiated cells for 7 days, and cDNA was generated with a HiScriptIIQ RT SuperMix for qPCR (Vazyme Biotech, Nanjing, China), followed by analysis with a ChamQTM SYBR Color qPCR Master Mix (Vazyme Biotech, Nanjing, China) (Vazyme Biotech). A LightCycler 480-II was used for amplification and detection (Roche, Mannheim, Germany). Supplementary Table S1 contains a list of primers. The target transcript’s levels were compared to those of the internal reference (2−ΔΔCT method).

PCSCs were cultured in a 6-well plate (3*10⁵ cells per well) for 24 h, followed by the treatment of IONPs (100 mg/ml) for 3 days. After that, total RNA was isolated using an RNA-Quick Purification Kit (Yishan Biotech, Shanghai, China) and cDNA was generated using a HiScript II Q RT SuperMix according to the manufacturer’s instructions. Finally, the quantitative PCR was detected using a ChamQTM SYBR Color qPCR Master Mix (Vazyme Biotech). Supplementary Table S1 listed the forward and reverse primers of the investigated osteogenic-related genes.