Detecting testis- and epididymis-specific methylated promoters in human cell-free seminal DNA by MeDIP-qPCR.
- Author:
Huang-Tao GUAN
1
;
Jin-Ming LÜ
2
;
Chun-Lin WU
1
;
Hong-Gang LI
1
;
Chang-Hong ZHU
1
;
Cheng-Liang XIONG
1
Author Information
- Publication Type:Journal Article
- MeSH: Adult; DNA; chemistry; DNA Methylation; Epididymis; metabolism; Epigenesis, Genetic; Humans; Male; Polymerase Chain Reaction; Promoter Regions, Genetic; Semen; chemistry; Testis; metabolism
- From: National Journal of Andrology 2013;19(11):977-983
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVETo establish a method of methyl-DNA immunoprecipitation (MeDIP)-real time quantitative PCR (qPCR) for detecting the promoter methylation level in cell-free seminal DNA (cfsDNA).
METHODSWe obtained cfsDNA samples from 6 normozoospermia men (the NZ group) and 6 post-vasectomy patients (the PV group), and mixed the samples from different individuals of each group, respectively. Then we made DNA fragments by ultrasonication, separated the methylated DNA fragments by MeDIP, and determined the methylation level of the promoters in cfsDNA by qPCR.
RESULTSThe methylation levels of the promoters PRAME, PEG10, MORC1, GML, HOXA5, DNMT3L, SNURF, MSH4, DAZ1 and CLPB were 14.93, 2.64, 0.69, 2.66, 17.50, 21.10, 5.98, 2.28, 13.50 and 3.86%, respectively, in the NZ group, obviously lower than 121.25, 73.62, 16.25, 42.90, 76.74, 112.40, 59.79, 25.85, 91.90 and 64.53% in the PV group. The results of MeDIP-qPCR for the methylation of PRAME, MORC1, GML, HOXA5, DNMT3L, SNURF, MSH4 and DAZ1 were coincident with the results of genome-wide promoter methylation microarray.
CONCLUSIONMeDIP-qPCR can quantitatively measure the promoter methylation level in cfsDNA, and effectively determine the testis- and epididymis-specific methylated promoters in human semen.
