A 5-mC Dot Blot Assay Quantifying the DNA Methylation Level of Chondrocyte Dedifferentiation In Vitro

Zhaofeng Jia; Yujie Liang; Bin Ma; Xiao Xu; Jianyi Xiong; Li Duan; Daping Wang

doi:10.3791/55565

A subscription to JoVE is required to view this content. Sign in or start your free trial.

Method Article

A 5-mC Dot Blot Assay Quantifying the DNA Methylation Level of Chondrocyte Dedifferentiation In Vitro

16.2K views

⸱

DOI:

10.3791/55565

⸱

May 17th, 2017

Zhaofeng Jia¹^,² , Yujie Liang³ , Bin Ma⁴^,⁵ , Xiao Xu²^,⁶ , Jianyi Xiong² , Li Duan² , Daping Wang¹^,²

¹Guangzhou Medical University, ²Shenzhen Key Laboratory of Tissue Engineering, Shenzhen Laboratory of Digital Orthopeadic Engineering, Department of Orthopedics, Shenzhen Second People's Hospital (The First Hospital Affiliated to Shenzhen University), ³Department of Chemistry, The Chinese University of Hong Kong, ⁴School of Biomedical Engineering, Shanghai Jiao Tong University, ⁵Renji Hospital Clinical Stem Cell Research Center, Shanghai Jiao Tong University School of Medicine, ⁶Shantou University Medical College

Summary

We present a method to quantify DNA methylation based on the 5-methylcytosine (5-mC) dot blot. We determined the 5-mC levels during chondrocyte dedifferentiation. This simple technique could be used to quickly determine the chondrocyte phenotype in ACI treatment.

Abstract

The dedifferentiation of hyaline chondrocytes into fibroblastic chondrocytes often accompanies monolayer expansion of chondrocytes in vitro. The global DNA methylation level of chondrocytes is considered to be a suitable biomarker for the loss of the chondrocyte phenotype. However, results based on different experimental methods can be inconsistent. Therefore, it is important to establish a precise, simple, and rapid method to quantify global DNA methylation levels during chondrocyte dedifferentiation.

Current genome-wide methylation analysis techniques largely rely on bisulfite genomic sequencing. Due to DNA degradation during bisulfite conversion, these methods typically require a large sample volume. Other methods used to quantify global DNA methylation levels include high-performance liquid chromatography (HPLC). However, HPLC requires complete digestion of genomic DNA. Additionally, the prohibitively high cost of HPLC instruments limits HPLC's wider application.

In this study, genomic DNA (gDNA) was extracted from human chondrocytes cultured with varying number of passages. The gDNA methylation level was detected using a methylation-specific dot blot assay. In this dot blot approach, a gDNA mixture containing the methylated DNA to be detected was spotted directly onto an N⁺ membrane as a dot inside a previously drawn circular template pattern. Compared with other gel electrophoresis-based blotting approaches and other complex blotting procedures, the dot blot method saves significant time. In addition, dot blots can detect overall DNA methylation level using a commercially available 5-mC antibody. We found that the DNA methylation level differed between the monolayer subcultures, and therefore could play a key role in chondrocyte dedifferentiation. The 5-mC dot blot is a reliable, simple, and rapid method to detect the general DNA methylation level to evaluate chondrocyte phenotype.

Introduction

Autologous chondrocyte implantation (ACI) is a relatively new, state-of-the-art procedure to treat articular cartilage defects¹^,². One of the crucial steps in ACI is amplification of chondrocytes via monolayer culture in vitro. During amplification, the hyaline chondrocytes easily lose their phenotype and become dedifferentiated, which is undesirable for ACI treatment³^,⁴. To optimize the outcome of ACI treatment, the extent of chondrocyte dedifferentiation should be determined before replantation. It is imperative to establish an economic and ....