Pikoreal96

First-strand cDNA was synthesized from total RNA using the PrimeScript RT Reagent Kit with gDNA Eraser (Takara) according to the manufacturer’s instructions. Quantitative real-time PCR (qRT-PCR) analysis was performed with the PikoReal 96 (Thermo, USA) using the AceQ qPCR SYBR Green Master Mix (Vazyme). The housekeeping As S7 gene was used as an endogenous control. The primers are shown in Supplementary Table S2.

The copy number of the T-DNA inserted into the rice genome was determined by qPCR using the PIKOREAL 96 (Thermo Fisher Scientific, Waltham, MA, United States) real-Time PCR system. Sixteen pCEiEPSPS transgenic lines and 11 pCP4-EPSPS transgenic lines were randomly selected for the copy number analysis. Quantitative PCR was performed in a 10 μL reaction mixture containing 5 μL of 2× SYBR Mixture, 0.25 mM of each primer, and 40 ng of the genomic DNA. The amplification procedure was 94°C for 7 min followed by 40 cycles of 94°C for 15 s, 55°C for 15 s, 72°C for 15 s (fluorescence detection), and 60°C for 30 s. The melt-curve analysis was performed to confirm that only one product has been amplified. The procedure was a 60–95°C cycle of being increased by 0.2°C and kept for 1 s each cycle. The copy number was estimated using the 2^–ΔΔCt method (Livak and Schmittgen, 2001 (link)). The single-copy endogenous gene SPS of rice was used as the internal reference gene (Yang et al., 2005 (link)). The qPCR primers for EiEPSPS, CP4-EPSPS, and SPS are EiEPSPSqPCR-F (5′-ATGCACACGCAAGACGTTCC-3′) and EiEPSPSqPCR-R (5′-CAGCAGCAGCTCTATGGCTGAG-3′), CP4-EPSPSqPCR-F (5′-AGCGTGGTCACTGCACAGAT-3′) and CP4-EPSPSqPCR-R (5′-GATGGCTGATGGACTTGTCG-3′) and SPSqPCR-F (5′-TTGCGCCTGAACGGATAT-3′) and SPSqPCR-R (5′-CGG TTGATCTTTTCGGGATG-3′), respectively.

The trizol was used to extract total RNA from cells and rat myocardial tissues. Primers of target genes (Table 1) were synthesized by Tsingke Biotechnology Co., Ltd. The cDNA was obtained by reverse transcription of mRNA using HiFscript cDNA Synthesis Kit (CW2569, CWBIO). A fluorescent quantitative PCR instrument (PIKOREAL96, Thermo) was used to amplify cDNA and detect the fluorescence signal. GAPDH was used as an internal reference. The relative expression levels of target genes were calculated by the 2^−ΔΔCt method.

Total RNA from human chondrocytes and rat chondrocytes was extracted using TRIzol^®. The RNA was reverse transcribed to cDNA by reverse transcription using an mRNA reverse transcription kit (CW2569, Cwbio) and miRNA reverse transcription kit (CW2141, Cwbio). Primers were designed and synthesized by Beijing Tsingke Biotechnology, and the sequences are listed in Table 1. Real-time Quantitative PCR (RT-qPCR) was performed using fluorescence quantitative PCR kit (PIKOREAL96, Thermo). The fluorescence intensity during the reaction was recorded in real time. The gene expression was calculated by 2^−ΔΔCt method compared with β-actin, U6, or 5S.

More specific details were described in the authors' previous studies.^[38] Specifically, Trizol reagent (TAKARA) were used to isolate the total mRNA from TS tissue samples or hPSCs. RNA concentration and quality were determined by NanoPhotometer (IMPLEN), and the final concentration of RNA was adjusted to 500 ng mL⁻¹. cDNA was produced from 1 mg RNA and then amplified using Ex Taq DNA polymerase (Takara) under the following reaction conditions: denaturation at 94 °C for 1 min, annealing at 58 °C for 45 s, and extension at 72 °C for 30 s; the cycle number was 35. The primer pairs used in this work are as listed in Table S1, Supporting Information. Real‐time PCR was performed on PikoReal 96 (Thermo Scientific).

The total RNA of kidney tissues and HRMCs in each group were isolated using TRIzol^® reagent (15596026; Thermo Fisher, Waltham, MA, USA). cDNAs were synthesized using mRNA reverse transcription kit (CW2569; CWBIO, Beijing, China). The PCR was performed using Ultra SYBR Mixture (CW2601; CWBIO). The fluorescent quantitative PCR system was Thermo Fisher (PIKOREAL96). The 2^−ΔΔCt method was applied to calculate the relative expression level. Actin and 5S were selected as internal reference mRNA and microRNA. The primer sequences (Shenggong, Shanghai, China) are listed in Table 2.