Normalizing gene expression levels in mouse fetal germ cells.
Real-time PCR has become a popular method to analyze transcription of genes that are developmentally regulated during organogenesis of the testes and ovaries. However, the heterogenous cell populations and commitment to strikingly different developmental pathways of the germ and somatic cells in these organs complicate analysis of this process. The selection of suitable reference genes for quantifying gene expression in this system is essential, but to date it has not been sufficiently addressed. To rectify this problem, we have used fluorescence-activated cell sorting to purify germ cells from mouse fetal testes and ovaries and examined 16 common housekeeping genes for their suitability as reference genes. In pure populations of germ cells isolated from Embryonic Day 12.5 (E12.5) to E15.5 male and female gonads, Mapk1 and Sdha were identified as the most stable reference genes. Analysis of the heterogenous fraction of gonadal somatic cells revealed that Canx and Top1 were stable in both sexes, whereas a comparative analysis of germ and somatic cell populations identified Canx and Mapk1 as suitable reference genes through these developmental stages. Application of these reference genes to quantification of gene expression in developing gonads revealed that past assays, which employed nonverified reference genes, have in some cases provided misleading gene expression profiles. This study has identified suitable reference genes to directly compare expression profiles of genes expressed in germ and somatic cells of male and female fetal gonads. Application of these reference genes to expression analysis in fetal germ and somatic cells provides a more accurate system in which to profile gene expression in these tissues.
van den Bergen
Jocelyn A
JA
Department of Paediatrics, Murdoch Children's Research Institute, Australian Research Council Centre of Excellence in Biotechnology and Development, University of Melbourne, Royal Children's Hospital, Melbourne, Victoria, Australia.
Miles
Denise C
DC
Sinclair
Andrew H
AH
Western
Patrick S
PS
D000818:Animals; D037281:Calnexin / Q000235:genetics / Q000378:metabolism; D002469:Cell Separation; D004264:DNA Topoisomerases, Type I / Q000235:genetics / Q000378:metabolism; D004622:Embryo, Mammalian / Q000378:metabolism*; D005260:Female; D005434:Flow Cytometry; D018507:Gene Expression Regulation, Developmental; D005854:Germ Cells / Q000378:metabolism*; D006066:Gonads / Q000196:embryology* / Q000378:metabolism; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D019950:Mitogen-Activated Protein Kinase 1 / Q000235:genetics / Q000378:metabolism; D038081:Organogenesis / Q000235:genetics*; D011247:Pregnancy; D012313:RNA / Q000302:isolation & purification / Q000378:metabolism / Q000592:standards; D020871:RNA Stability; D016372:RNA, Antisense / Q000096:biosynthesis / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000592:standards*; D012737:Sex Factors
D016372:RNA, Antisense; D037281:Calnexin; D012313:RNA; C535148:Mapk1 protein, mouse; D019950:Mitogen-Activated Protein Kinase 1; D004264:DNA Topoisomerases, Type I