Comparison of three different techniques of human sperm DNA isolation for methylation assay.
10.1007/s11596-015-1532-0
- Author:
Hong-fang YUAN
1
;
Martin KUETE
2
;
Li SU
3
;
Fan YANG
4
;
Zhi-yong HU
4
;
Bo-zhen TIAN
4
;
Hui-ping ZHANG
4
;
Kai ZHAO
5
,
6
Author Information
1. Center of Reproductive Medicine, Institute of Family Planning Research, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, China. 350358685@qq.com.
2. Center of Reproductive Medicine, Institute of Family Planning Research, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, China. martinezkuete@yahoo.fr.
3. Family Planning Women and Children Service Center of ZengDu District, Suizhou, 441300, China.
4. Center of Reproductive Medicine, Institute of Family Planning Research, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, China.
5. Center of Reproductive Medicine, Institute of Family Planning Research, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, China. kevingull@
6. com.
- Publication Type:Journal Article
- Keywords:
TianGen kit;
human sperm DNA;
isolation;
modified guanidinium thiocyanate;
phenol-chloroform
- MeSH:
Adult;
DNA;
isolation & purification;
metabolism;
DNA Methylation;
Electrophoresis, Agar Gel;
Humans;
Male;
Spermatozoa;
metabolism
- From:
Journal of Huazhong University of Science and Technology (Medical Sciences)
2015;35(6):938-942
- CountryChina
- Language:English
-
Abstract:
Human sperm DNA is an important genetic and epigenetic material, whose chromatin structure differs from that of somatic cells. As such, conventional methods for DNA extraction of somatic cells may not be suitable for obtaining sperm DNA. In this study, we evaluated and compared three sperm DNA extraction techniques, namely, modified guanidinium thiocyanate method (method A), traditional phenol-chloroform method (method B), and TianGen kit method (method C). Spectrophotometry and agarose gel electrophoresis analyses showed that method A produced DNA with higher quantity and purity than those of methods B and C (P<0.01). PCR results revealed that method A was more reliable in amplifying DEAD-box polypeptide 4 (DDX4) and copy number variations (CNVs) than methods B and C, which generated false-positive errors. The results of sperm DNA methylation assay further indicated that methods A and B were effective, and the former yielded higher quantitative accuracy. In conclusion, the modified guanidinium thiocyanate method provided high quality and reliable results and could be an optimal technique for extracting sperm DNA for methylation assay.
