title: Reference gene selection for gene expression analysis of oocytes collected from dairy cattle and buffaloes during winter and summer.
abstract: Oocytes from dairy cattle and buffaloes have severely compromised developmental competence during summer. While analysis of gene expression is a powerful technique for understanding the factors affecting developmental hindrance in oocytes, analysis by real-time reverse transcription PCR (RT-PCR) relies on the correct normalization by reference genes showing stable expression. Furthermore, several studies have found that genes commonly used as reference standards do not behave as expected depending on cell type and experimental design. Hence, it is recommended to evaluate expression stability of candidate reference genes for a specific experimental condition before employing them as internal controls. In acknowledgment of the importance of seasonal effects on oocyte gene expression, the aim of this study was to evaluate the stability of expression levels of ten well-known reference genes (ACTB, GAPDH, GUSB, HIST1H2AG, HPRT1, PPIA, RPL15, SDHA, TBP and YWHAZ) using oocytes collected from different categories of dairy cattle and buffaloes during winter and summer. A normalization factor was provided for cattle (RPL15, PPIA and GUSB) and buffaloes (YWHAZ, GUSB and GAPDH) based on the expression of the three most stable reference genes in each species. Normalization of non-reference target genes by these reference genes was shown to be considerably different from normalization by less stable reference genes, further highlighting the need for careful selection of internal controls. Therefore, due to the high variability of reference genes among experimental groups, we conclude that data normalized by internal controls can be misleading and should be compared to not normalized data or to data normalized by an external control in order to better interpret the biological relevance of gene expression analysis.
authors.affiliation: Departamento de Genética e Evolução, Centro de Ciências Biológicas e da Saúde, Universidade Federal de São Carlos, São Carlos, SP, Brazil; Departamento de Medicina Veterinária, Faculdade de Zootecnia e Engenharia de Alimentos, Universidade de São Paulo, Pirassununga, SP, Brazil.
authors.forename: Carolina Habermann
authors.initials: CH
authors.lastname: Macabelli
authors.affiliation: Departamento de Medicina Veterinária, Faculdade de Zootecnia e Engenharia de Alimentos, Universidade de São Paulo, Pirassununga, SP, Brazil; Departamento de Reprodução Animal, Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo, São Paulo, SP, Brazil.
authors.forename: Roberta Machado
authors.initials: RM
authors.lastname: Ferreira
authors.affiliation: Departamento de Medicina Veterinária Preventiva e Reprodução Animal, Faculdade de Ciências Agrárias e Veterinárias, Universidade Estadual Paulista, Jaboticabal, SP, Brazil.
authors.forename: Lindsay Unno
authors.initials: LU
authors.lastname: Gimenes
authors.affiliation: Unidade de Pesquisa e Desenvolvimento de Registro, Pólo Regional Vale do Ribeira-APTA, Registro, SP, Brazil.
authors.forename: Nelcio Antonio Tonizza
authors.initials: NA
authors.lastname: de Carvalho
authors.affiliation: Departamento de Reprodução Animal, Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo, São Paulo, SP, Brazil.
authors.forename: Júlia Gleyci
authors.initials: JG
authors.lastname: Soares
authors.affiliation: Departamento de Reprodução Animal, Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo, São Paulo, SP, Brazil; MSD Saúde Animal, São Paulo, SP, Brazil.
authors.forename: Henderson
authors.initials: H
authors.lastname: Ayres
authors.affiliation: Vida Reprodutiva Consultoria, Cravinhos, SP, Brazil.
authors.forename: Márcio Leão
authors.initials: ML
authors.lastname: Ferraz
authors.affiliation: WTA Tecnologia Aplicada LTDA, Cravinhos, SP, Brazil.
authors.forename: Yeda Fumie
authors.initials: YF
authors.lastname: Watanabe
authors.affiliation: WTA Tecnologia Aplicada LTDA, Cravinhos, SP, Brazil.
authors.forename: Osnir Yoshime
authors.initials: OY
authors.lastname: Watanabe
authors.affiliation: Departamento de Medicina Veterinária, Faculdade de Zootecnia e Engenharia de Alimentos, Universidade de São Paulo, Pirassununga, SP, Brazil; Departamento de Cirurgia, Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo, São Paulo, SP, Brazil.
authors.forename: Juliano Rodrigues
authors.initials: JR
authors.lastname: Sangalli
authors.affiliation: Centre de recherche em reproduction animale, Faculté de medicine vétérinaire, Université de Montréal, St. Hyacinthe, QC, Canada.
authors.forename: Lawrence Charles
authors.initials: LC
authors.lastname: Smith
authors.affiliation: Departamento de Reprodução Animal, Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo, São Paulo, SP, Brazil.
authors.forename: Pietro Sampaio
authors.initials: PS
authors.lastname: Baruselli
authors.affiliation: Departamento de Medicina Veterinária, Faculdade de Zootecnia e Engenharia de Alimentos, Universidade de São Paulo, Pirassununga, SP, Brazil; Departamento de Cirurgia, Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo, São Paulo, SP, Brazil.
authors.forename: Flávio Vieira
authors.initials: FV
authors.lastname: Meirelles
authors.affiliation: Departamento de Genética e Evolução, Centro de Ciências Biológicas e da Saúde, Universidade Federal de São Carlos, São Carlos, SP, Brazil; Departamento de Medicina Veterinária, Faculdade de Zootecnia e Engenharia de Alimentos, Universidade de São Paulo, Pirassununga, SP, Brazil; Departamento de Cirurgia, Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo, São Paulo, SP, Brazil.
authors.forename: Marcos Roberto
authors.initials: MR
authors.lastname: Chiaratti
mesh_terms: D000818:Animals; D002020:Buffaloes; D002417:Cattle; D003612:Dairying; D005260:Female; D015870:Gene Expression; D020869:Gene Expression Profiling; D020043:Genes, Essential; D009865:Oocytes / Q000166:cytology / Q000378:metabolism*; D060888:Real-Time Polymerase Chain Reaction / Q000592:standards*; D012015:Reference Standards; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000592:standards*; D012621:Seasons