All in one mastermix kit

The cellular RNA was extracted using Trizol reagent (Invitrogen) and reverse transcribed into cDNA by Quantscript RT kit (abm, Richmond, BC, Canada). KCNK6 specific primers were used for real-time quantitative polymerase chain reaction (PCR). A 5 × All-In-OneMasterMix kit (abm, Richmond, BC, Canada) was used for real-time quantitative PCR. A CFX96 real-time PCR detection system (Bio-Rad, Hercules, CA, United States) was used to detect the relative expression level of KCNK6. The sequence of qPCR primers was as follows: KCNK6-F, 5′-CTAAACCCCTCCTGTGTGCT-3′; KCNK6-R, 5′-CAACACCTCACCTCCTCCAT-3′; GAPDH-F, 5′-CAACGGATTTGGTCGTATTGG-3′; and GAPDH-R, 5′-TGACGGTGCCATGGA ATTT-3′.

Total RNA was extracted from human samples or mouse primary chondrocytes using TRIzol reagent (Invitrogen) and then reverse transcribed into cDNA using a 5X All-In-One MasterMix kit (ABM). qPCR was performed using iTaq Universal SYBR Green Supermix (Bio-Rad) on a CFX96 real-time system (Bio-Rad). The PCR primers used included hACTB (NM_001101.5): 5’-ATT GGC AAT GAG CGG TTC-3’ and 5’-GGA TGC CAC AGG ACT CCA-3’; hFAP (NM_001291807.3): 5’-TGG CGA TGA ACA ATA TCC TAG A-3’ and 5’-ATC CGA ACA ACG GGA TTC TT-3’; hOLN/CLEC11A (NM_002975.3): 5’-GAG AGG GAG GCC CTG ATG-3’ and 5’-AAC AGT TCC GGC AGG ATT C-3’; mActb (NM_007393.5): 5’-GCT CTT TTC CAG CCT TCC TT-3’ and 5’-CTT CTG CAT CCT GTC AGC AA-3’; mAcan (NM_007424.3): 5’-TGA AGC AGA AGG TCT GGA CA-3’ and 5’-CCA GAA GGA ATC CCA CTA ACA-3’; mCol2a1 (NM_031163.3): 5’-GTC CCC CTG GCC TTA GTG-3’ and 5’-CCA CCA GCC TTC TCG TCA-3’; mMmp3 (NM_010809.2): 5’-TGC AGC TCT ACT TTG TTC TTT GA-3’ and 5’-AGA GAT TTG CGC CAA AAG TG-3’; mOln (NM_009131.3): 5’-AGG TCC TGG GAG GGA GTG-3’ and 5’-GGG CCT CCT GGA GAT TCT T-3’.

To verify the reliability of RNA-seq data, DEGs of candidate resistance genes were selected for quantitative real-time polymerase chain reaction (qRT-PCR). Total RNA was extracted as described above. cDNA was synthesized using the 5X All-In-One MasterMix Kit (abm, Vancouver, BC, Canada), which was used as a template for qRT-PCR. The qRT-PCR was performed with various primers (Supplementary Table 1) on a qTOWER 2.2 system (Analytik Jena AG, Jena, Germany). Reactions were prepared using the SYBR Premix Dimer Eraser kit (Takara) according to the manufacturer’s instructions. Thermal cycler settings included denaturation at 95°C for 2 min, followed by 40 cycles of 95°C for 10 s and 60°C for 34 s. The samples were analyzed in a triplicate. The 2^–ΔΔCt method was employed to calculate the relative gene expression levels and data were analyzed with the Student’s t-test (p < 0.05) using SPSS (IBM, Armonk, NY, United States) (Livak and Schmittgen, 2001 (link)). β-Actin and GAPDH genes were used as reference to normalize quantification of the target gene expression.

Utilizing the NCBI gene bank, primers of 7 randomly selected DEGs, three different isoforms of MyHC genes (MyHC I, IIA, and IIB) and the house-keeping gene GAPDH were designed by Primer Premier 5 software (Table S1). Primers were synthesized by Hangzhou Youkang Biotechnology Co., Ltd. (Zhejiang, China). Total RNA from cells or tissues was used to synthesize cDNA by a 5X All-In-One MasterMix Kit (abm, China). The cDNA was used as a template for quantitative PCR by using the EvaGreen 2X qPCR MasterMix Kit (abm, China). There were three replicates for each sample. The relative expression level of each related gene was calculated using the 2^-△△CT statistical analysis method.

Total RNA from human samples or cells was extracted using the TRIzol reagent (Invitrogen) and quantified by a NanoDrop spectrophotometer. Complementary cDNA was synthesized from total RNA using a 5X All-In-One MasterMix kit (ABM). This system utilized SYBR Green dye to detect and quantify the amplification of specific target genes. The obtained data were normalized to GAPDH to determine relative gene expression levels, which serves as an internal control. The fold change between samples or experimental conditions was calculated by the 2^−∆∆Ct method. Primer sequences used in our study are shown in Supplementary Table S1.