Methylation-specific PCR (MSP) assays
MSP assays were performed to assess the DNA methylation status.
Briefly, genomic DNA was extracted from placentas and HTR-8/SVneo cells
via DNA extraction kits (TIANGEN, Beijing, China) following the
manufacturer’s instructions. The bisulfite conversion of DNA was carried
out via EZ DNA methylation Gold (ZYMO Research Corporation, USA).
Methylation-specific primers were subsequently designed to selectively
amplify either methylated or unmethylated DNA sequences of the TP53BP2
promoter region. After amplification, the PCR products were analyzed via
2% gel electrophoresis to visualize the presence or absence of
specific bands corresponding to methylated or unmethylated DNA,
respectively. The primer sequences for the MSP assays are provided in
Table S2.