Rnaeasy animal rna extraction kit

The total RNA of BMSCs was extracted by RNAeasy™ Animal RNA Extraction Kit (R0026, Beyotime Biotechnology) according to the manufactures recommended procedure. After determining the concentration, reverse transcription was performed using the Evo M-MLV RT Kit (AG11728, Accurate Biology). The real-time qPCR process was done on the ABI ViiA 7(Life Technologies) with the SYBR Green Pro Taq HS Kit (AG11701, Accurate Biology) following the recommended protocol.

Mouse articular chondrocytes were obtained from Wuhan Procell Life Science and Technology Co., Ltd. The cells were cultured in DMEM/F12 medium supplemented with 10% fetal bovine serum. For the inflammation model, the cells were stimulated with IL-1β at a concentration of 10 ng/mL for 24 h. Total RNA was extracted using the RNAeasy™ Animal RNA Extraction Kit (Beyotime Biotechnology, China), and the quality was assessed through spectrophotometry using the NanoDrop One instrument. Subsequently, reverse transcription was performed to generate cDNA. The extracted RNA was reverse transcribed into cDNA using the PrimeScript™ RT Master Mix (TAKARA, Japan). The cDNA was amplified using the PowerUp™ SYBR^® Green Master Mix (Applied Biosystems, USA) on the ABI 7500 Real-Time PCR System. RT-qPCR was conducted to analyze the expression of different genes, with Gapdh serving as an internal reference. The relative mRNA expression levels were calculated using the 2^(-ΔΔCt) method, and statistical significance was determined by a p-value less than 0.05. Please refer to Table 1 for the primer sequences.

Total RNA was extracted from the collected BLCA tumor tissues and adjacent normal tissues using the RNAeasy™ Animal RNA Extraction Kit (Beyotime). Subsequently, the reverse transcription process was performed using the PrimeScript™ RT reagent Kit (Takara), and qRT-PCR was conducted with the TB Green® Premix Ex Taq™ II Kit (Takara), following the manufacturer's instructions. The primer sequences are shown in Table 1.
Table 1

The list of the primers used for qRT-PCR.

Gene symbol	Forward or reverse primer	Primer sequence (5'–3')
GAPDH	Forward	GGAAGCTTGTCATCAATGGAAATC
	Reverse	TGATGACCCTTTTGGCTCCC
NRTN	Forward	ACCCTGGACGCCCGGATT
	Reverse	CGCAGTAGCGGAACAGCACC
MAP3K8	Forward	TCGCTCAGCCTATCCCTCCTA
	Reverse	GTTCCAGCTCCTTCCTACTCAG
RAC3	Forward	CTCCTACCCCCAAACTGACG
	Reverse	TTCACAGAGCCCACCAATCTC
PDGFD	Forward	GGTGAAAGGAAACGGCTACG
	Reverse	CTCTAATAATGGTACTGGTTTCGGA
JUN	Forward	TGGGTGCCAACTCATGCTAA
	Reverse	TTCTTCGTTGCCCCTCAGC
MAP3K20	Forward	GTTAGATACTCTGAGGATGCGGC
	Reverse	GTTGATACTTAATGGGCACCTGG
IGF1	Forward	GGTGGATGCTCTTCAGTTCGT
	Reverse	GCAATACATCTCCAGCCTCCTTA
PTPRR	Forward	GCAGGAATAGGTAGAACAGGGTG
	Reverse	GCACCATTCCACCTCTATCCA
DUSP2	Forward	TGCTGTCCCGATCTGTGCT
	Reverse	CAGGAACAGGTAGGGCAAGA
PDGFRA	Forward	CTTTGGATTGAACCCTGCTGA
	Reverse	GACATCTCGTGCCAACTCCA

The feasibility of the RPA-CRISPR/Cas12a assay for detecting SVA was assessed using 85 clinical specimens. These swine specimens were collected from pig tissues with vesicular lesions in Henan Province between April 2019 and February 2023. These specimen were collected for laboratory diagnosis, and not specifically for our study. Furthermore, no animals were experimentally infected in our study; no IACUC protocols apply or are available. Tissue homogenate was prepared as a 10% (w/v) suspension using Minimum Essential Medium (ThermoFisher Scientific, MA, USA) and clarified by centrifugation for 10 min at 12000r/s. The supernatant was collected. The nucleic acids of the samples were isolated by RNAeasy™ Animal RNA Extraction Kit (Beyotime, Shanghai, China) and tested by the RPA-CRISPR/Cas12a assay. The samples were also analyzed by a RT-qPCR assay targeting the capsid protein gene according to the China Agricultural industry standard (NY/T 3790-2020). In both of these detection methods, positive and negative controls were set up separately. Prism 5.0 software (GraphPad) was used to analyze the correlation between RPA-CRISPR/Cas12a threshold time (TT) and RT-qPCR cycle threshold (Ct) values.

After treatment with QUCO-1 for 24 h, the RKO cells were washed three times with cold PBS and the mitochondria were extracted using a cell mitochondrial isolation kit (beyotime, C3601). RNA and DNA extractions were performed according to the instructions of the RNAeasy™ animal RNA extraction kit (beyotime, R0027) and the genomic DNA small extraction kit (beyotime, D0063). Finally, their concentrations were determined using trace ultraviolet light after extraction.

We obtained rat articular chondrocytes from Wuhan Procell Life Science and Technology Co., Ltd. Cells were cultured using DMEM/F12 medium containing 10% fetal bovine serum. The inflammation model group was stimulated with a medium containing IL-1β at a concentration of 10 ng/ml for 24 h. Total RNA was extracted with RNAeasy™ Animal RNA Extraction Kit (Beyotime Biotechnology, China), and quality control was conducted by NanoDrop one spectrophotometer. Then, reverse transcription was performed to produce cDNA. The extracted RNA was reverse transcribed to cDNA using PrimeScript™ RT Master Mix (TAKARA, Japan). cDNA was extracted using PowerUp™ SYBR® Green Master Mix (Applied Biosystems, USA) and ABI 7500Real-TimePCR System. RT-qPCR was performed to detect the expression of differential genes. GAPDH was used as an internal reference. The relative mRNA expression level was calculated with the 2^−ΔΔCt method, and P < 0.05 indicated a significant difference. The primer sequences are shown in Table 1.