Ezscript reverse transcription mix 2 kit

After 7 and 14 days of chondrogenic differentiation, total RNA was isolated and complementary DNA (cDNA) was prepared using the RNA purification kit and 4 × EZscript Reverse Transcription Mix II kit (EZBioscience, Roseville, MN, USA) according to the manufacturer’s instructions. Real-time quantitative PCR was performed using the SYBR Green Master Mix (EZBioscience, Roseville, MN, USA) in a real-time PCR System (QuantStudio™ 7 Flex, Thermo Fisher Scientific, Waltham, MA, USA). The reaction was performed with a total volume of 10 μL in each reaction well under the following conditions: 95 °C for 5 min to activate, followed by 40 cycles of 10 s at 95 °C and 60 s at 60 °C. The quantitative gene expression was normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and the relative expression was determined using the ΔΔCt method. Each sample has three technical replicates. Primer sequences are listed in Table 2.

Total RNA was extracted from the cultured cells by using TRIZol reagent (Tsingke, Beijing, China). The cDNA was synthesized with 2 μg of RNA using the 4×EZscript Reverse Transcription Mix II Kit (EZBioscience, Roseville, MN, USA). Then, each 96-well plate well was mixed with 6.5 μL of cDNA (diluted at 1:50), 7.5 μL of qPCR SYBR Mix (Tsingke, Beijing, China), and 1 μL of primers (10 mM). The primers used in the study are listed in Table S6.