Quantstudio 6 and 7 flex software

RNA was isolated using RNeasy Mini Kit (Qiagen Cat# 74104) protocol and eluted in a volume of 40uL of RNase free water. Real-time quantitative reverse transcription PCR was performed using TaqMan 1-step RNA to Ct (Thermo Cat# 4392938) with CDC primer/probe kit (IDT − 10006713) against the N1 gene. Samples were analyzed using Viia7 (ThermoFisher) along with Quantstudio 6 and 7 Flex software (ThermoFisher). Genomes/mL were interpolated using Ct values and genomic standard (BEI - NR-52358) run in triplicate.

Total RNA was extracted from differentiated chondrogensis respectively cultured in control serum and serum containing BSHXF using TRIzol (Invitrogen, CA, USA), and cDNA was generated from 2 μg RNA using RevertAid First Strand cDNA Synthesis Kit (Invitrogen, CA, USA) following the manufacturer’s instruction. Real-time PCR was performed for target gene using the SYBR Premix Ex Taq™ II (Takara, Dalian, China) with a QuantStudio™ 7 Flex Real-Time PCR System (Thermo Scientific, MA, USA) according to the manufacturer’s instruction. The specific primers are as followed: primer sequences of Mmp13, forward primer 5′-TTTGAGAACACGGGGAAGA-3′ and reverse primer 5′-ACTTTGTTGCCAATTCCAGG-3′; primer sequences of Actb, forward primer 5′-GGAGATTACTGCCCTGGCTCCTA-3′ and reverse primer 5′-GACTCATCGTACTCCTGCTTGCTG-3′. Relative Mmp13 gene expression values were analysed using the comparative 2^−∆∆Ct method for relative quantification, which is implemented in the QuantStudio™ 6 and 7 Flex Software (Thermo Scientific, MA, USA). Actb was used as an endogenous control gene for PCR normalization concerning the amount of RNA added to the reverse transcription reactions.