一种检测衰老干细胞中干性相关基因CpG位点DNA甲基化变化的方案。

A Protocol for Detecting DNA Methylation Changes at CpG Sites of Stemness-Related Genes in Aging Stem Cells.

作者信息

Hodjat Mahshid, Daghighi Seyed Mojtaba, Abdollahi Mohammad

机构信息

Dental Research Center, Dentistry Research Institute, Tehran University of Medical Sciences, Tehran, Iran.

Department of Toxicology and Pharmacology, School of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran.

出版信息

Methods Mol Biol. 2025;2960:139-146. doi: 10.1007/7651_2024_590.

DOI:10.1007/7651_2024_590

PMID:39821804

Abstract

Aging adversely affects the self-renewal and differentiation capabilities of stem cells, which impairs tissue regeneration as well as the homeostasis. Epigenetic mechanisms, specifically DNA methylation, play a key role in the maintenance of pluripotency in stem cells and regulation of pluripotency-related gene expression. Age-related modifications in methylation patterns could influence the expression of genes critical for stem cell potency maintenance, including transcription factors Nanog and Sox2. The following chapter describes a step-by-step bisulfite sequencing protocol for detection of methylation changes in the aging stem cells and provides valuable insights into the stem cells epigenetic profile. Further, the methodology describes the steps of genomic DNA extraction, bisulfite conversion, real-time PCR amplification, and sequencing for an in-depth view of the epigenetic profile derived from aging stem cells.

摘要

衰老对干细胞的自我更新和分化能力产生不利影响，进而损害组织再生以及体内平衡。表观遗传机制，特别是DNA甲基化，在干细胞多能性的维持以及多能性相关基因表达的调控中起着关键作用。甲基化模式的年龄相关变化可能会影响对干细胞潜能维持至关重要的基因的表达，包括转录因子Nanog和Sox2。以下章节介绍了一种用于检测衰老干细胞中甲基化变化的亚硫酸氢盐测序分步方案，并为干细胞表观遗传特征提供了有价值的见解。此外，该方法还描述了基因组DNA提取、亚硫酸氢盐转化、实时PCR扩增和测序的步骤，以便深入了解衰老干细胞的表观遗传特征。