Sadegh Baradaran Mahdavi
1,2,3 
, Seyed Morteza Javadirad
4* , Mahsa Rafieian
2,3 , Parnian Poursafa
2 , Vajihe Azimian Zavareh
3 , Seyede Shahrbanoo Daniali
2 , Motahar Heidari-Beni
2 , Masoomeh Goodarzi-Khoigani
2 , Babak Vahdatpour
1 , Hossein Mirhendi
3,5 , Roya Kelishadi
2* 1 Department of Physical Medicine and Rehabilitation, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran
2 Child Growth and Development Research Center, Research Institute for Primordial Prevention of Non-Communicable Disease, Isfahan University of Medical Sciences, Isfahan, Iran
3 Core Research Facilities (CRF), Isfahan University of Medical Sciences, Isfahan, Iran
4 Department of Cell and Molecular Biology, Faculty of Biological Science and Technology, University of Isfahan, Isfahan, Iran
5 Department of Medical Parasitology and Mycology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran
Abstract
Introduction: It is believed that DNA methylation can modify disease susceptibility in response to environmental factors as early as the perinatal period. In this study, we aimed to present a streamlined DNA methylation analysis procedure for osteoporosis-related gene promoters in the umbilical cord blood.
Methods: The Prospective Epidemiological Research Studies in Iran (PERSIAN) birth cohort was established in 2016. In this study, a total of 300 umbilical cord blood samples were collected at the time of delivery. For all samples, DNA was extracted and converted using sodium bisulfite. Multiple primer sets were designed for Wnt1, Wnt10b, β-catenin, OPG, and RANKL gene promoters in the online MethPrimer platform. Next, bisulfite sequencing PCR (BSP), as the gold standard method for exploring methylated and unmethylated cytosines, was performed in a gradient-controlled setting. The PCR products were then purified and directly sequenced. Subsequently, the chromatograms were interpreted.
Results: For Wnt10b, β-catenin, and OPG genes, the converted DNA could be successfully amplified. The frequency of acceptable chromatograms for analysis was 195 for Wnt10b (195/300, 0.65%), 198 for β-catenin (198/300, 0.66%), and 50 for OPG (50/50, 100%).
Conclusion: BSP can be efficiently used to investigate the methylation of target gene promoters in umbilical cord blood DNA.