Sybr premix

The odontogenic differentiation-related gene expression was detected using Real-time PCR. SHEDs served as the blank control group, SHEDs/HA/TCP served as the negative control group, and SHEDs/hTDM and SHEDs/pTDM served as the experimental groups. RNAiso Plus (Takara Biomedical Technology, Beijing, China), PrimeScript™ RT reagent (Takara Biomedical Technology, Beijing, China), and SYBR Premix (QIAGEN, Hilden, Germany) were used to extract total RNA, reverse transcribe RNA to cDNA, and detect the related genes’ expression levels (DSPP, POSTN, TGF-β1, ALP, COL-1, OCN, and RUNX2). According to the instructions, the configuration of each 10 μL PCR reaction solution contained 5 μL SYBR Premix, 0.4 μL PCR forward primer, 0.4 μL PCR reverse primer, 1 μL DNA template, and 3.2 μL sterile water. After normalization to the GAPDH internal reference gene, relative mRNA expression was determined using the 2^−ΔΔCT method. Supplemental Table 1 displays Real-time PCR primer sequences. The experiment was repeated at least three times using biological samples and technical means.

For qRT-PCR, total RNA was prepared using the TRIzol reagent (Tel-Test, Inc., USA), and cDNA was synthesized using the SuperScript pre-amplification system (Invitrogen). The PCR primers and cycling conditions are described in the Xenopus Molecular Marker Resource (University of Texas). Additional primers are described in Table 2. The PCR reactions were performed with SYBR Premix (Qiagen, USA) and a thermal cycler real-time system (Qiagen Rotor-Gene-Q, USA).