Light cycler apparatus

The mRNA transcript levels of chondrogenic-related genes (Acan, Col2a1, and Sox9), as well as the inflammation related gene (Mmp13) within mouse chondrocytes cultured on HA-coated plates verses control were assessed by real-time PCR. At day 3, cells were harvested and lysed in Trizol (Invitrogen Inc., Carlsbad, CA, USA); mRNA was isolated according to the manufacturer's method. ReverTra Ace qPCR Master Mix kit (TOYOBO, Japan) and SYBR Green QPCR Master Mix (Takara) were used to perform reverse transcription and PCR, respectively, with a Light Cycler apparatus (Bio-rad, CFX-Touch). The relative expression level of target genes was then computed using the 2−ΔΔCt method. Each qPCR was performed on at least 3 distinct samples and results were illustrated as target gene expression normalized to the reference gene GAPDH.

Total RNA from the mouse efferent ductules was extracted using a standard TRIzol RNA isolation method (Invitrogen, Carlsbad, CA) as previously described (Wang et al., 2014 (link)). The reverse transcription and PCR experiments were performed with the Revertra Ace qPCR RT Kit (TOYOBO FSQ-101) using 0.5 μg of each sample, according to the manufacturer’s protocols. The quantitative real-time PCR was conducted in the LightCycler apparatus (Bio-Rad) using the FastStart Universal SYBR Green Master (Roche). The qPCR protocol was as follows: 95°C for 10 min; 40 cycles of 95°C for 15 s and 60°C for 1 min; and then increasing temperatures from 65°C to 95°C at 0.1 °C/s. The mRNA level was normalized to GAPDH in the same sample and then compared with the control. All primers are listed in Supplementary file 1 and Supplementary file 2.

To evaluate the mRNA transcript levels of chondrocytes specific genes (Col2a1, Aggrecan, Sox9, Col10a1, Mmp13, and Ihh), stem cell-specific genes (Oct4, Nanog, and Sox2), and three germ layer genes (Nestin, Map 2, Desmin, Msx1, and Sox17), chondrocyte spheres, iPSCs, and EBs were processed for total RNA extraction by using an RNAprep Micro Kit (TaKaRa, Japan) at 14 days or 7 days. The concentration of RNA was determined with an RNA analyzer (Quawell, USA). The cDNA was prepared with PrimeScript RT Master Mix (TaKaRa, Japan). RT-PCR was performed by using SYBR Green QPCR Master Mix (TaKaRa, Japan) with a Light Cycler apparatus (Bio-Rad, USA). Cycle conditions were as follows: activation of HotStarTaq DNA polymerase/inactivation of reverse transcriptase at 95 °C for 30 s; and 39 cycles of 95 °C for 5 s, and 60 °C for 30 s. The relative expression level of each target gene was calculated by using the 2^−ΔΔCt method. All of the primers’ information was provided in Table. S1. Results were repeated for three independent biological replicates. The RT-PCR data were statistically present as mean ± SEM and p values of significance were calculated by Student’s t test (tails = 2, Two-sample unequal variance) in Excel: *p < 0.05, **p < 0.01, ***p < 0.001, ns is no significance with p > 0.05.