title: Comparison of three different techniques of human sperm DNA isolation for methylation assay.
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.
authors.affiliation: Center of Reproductive Medicine, Institute of Family Planning Research, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, China.
authors.forename: Hong-Fang
authors.initials: HF
authors.lastname: Yuan
authors.affiliation: Center of Reproductive Medicine, Institute of Family Planning Research, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, China.
authors.forename: Martin
authors.initials: M
authors.lastname: Kuete
authors.affiliation: Family Planning Women and Children Service Center of ZengDu District, Suizhou, 441300, China.
authors.forename: Li
authors.initials: L
authors.lastname: Su
authors.affiliation: Center of Reproductive Medicine, Institute of Family Planning Research, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, China.
authors.forename: Fan
authors.initials: F
authors.lastname: Yang
authors.affiliation: Center of Reproductive Medicine, Institute of Family Planning Research, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, China.
authors.forename: Zhi-Yong
authors.initials: ZY
authors.lastname: Hu
authors.affiliation: Center of Reproductive Medicine, Institute of Family Planning Research, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, China.
authors.forename: Bo-Zhen
authors.initials: BZ
authors.lastname: Tian
authors.affiliation: Center of Reproductive Medicine, Institute of Family Planning Research, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, China.
authors.forename: Hui-Ping
authors.initials: HP
authors.lastname: Zhang
authors.affiliation: Center of Reproductive Medicine, Institute of Family Planning Research, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, China. kevingull@163.com.
authors.forename: Kai
authors.initials: K
authors.lastname: Zhao
mesh_terms: D000328:Adult; D004247:DNA / Q000302:isolation & purification / Q000378:metabolism*; D019175:DNA Methylation; D004587:Electrophoresis, Agar Gel; D006801:Humans; D008297:Male; D013094:Spermatozoa / Q000378:metabolism*
chemical_list: D004247:DNA
keywords: TianGen kit; human sperm DNA; isolation; modified guanidinium thiocyanate; phenol-chloroform