Reference Gene Selection for Gene Expression Analysis of Oocytes Collected from Dairy Cattle and Buffaloes during Winter and Summer

Author(s):Show less -

Macabelli, Carolina Habermann ^{[1, 2]} ; Ferreira, Roberta Machado ^{[1, 3]} ; Gimenes, Lindsay Unno ^[4] ; Tonizza de Carvalho, Nelcio Antonio ^[5] ; Soares, Julia Gleyci ^[3] ; Ayres, Henderson ^{[3, 6]} ; Ferraz, Marcio Leao ^[7] ; Watanabe, Yeda Fumie ^[8] ; Watanabe, Osnir Yoshime ^[8] ; Sangalli, Juliano Rodrigues ^{[1, 9]} ; Smith, Lawrence Charles ^[10] ; Baruselli, Pietro Sampaio ^[3] ; Meirelles, Flavio Vieira ^{[1, 9]} ; Chiaratti, Marcos Roberto ^{[1, 9, 2]}

Affiliation

^[1] Univ Sao Paulo, Fac Zootecnia & Engn Alimentos, Dept Med Vet, Pirassununga, SP - Brazil

^[2] Univ Fed Sao Carlos, Ctr Ciencias Biol & Saude, Dept Genet & Evolucao, BR-13560 Sao Carlos, SP - Brazil

^[3] Univ Sao Paulo, Fac Med Vet & Zootecnia, Dept Reprod Anim, Sao Paulo - Brazil

^[4] Univ Estadual Paulista, Fac Ciencias Agr & Vet, Dept Reprod Anim, Jaboticabal, SP - Brazil

^[5] APTA, Unidade Pesquisa Desenvolvimento Registro, Registro, SP - Brazil

^[6] Vida Reprod Consultoria, Cravinhos, SP - Brazil

^[7] MSD Saude Anim, Sao Paulo - Brazil

^[8] LTDA, WTA Tecnol Aplicada, Cravinhos, SP - Brazil

^[9] Univ Sao Paulo, Fac Med Vet & Zootecnia, Dept Cirurgia, Sao Paulo - Brazil

^[10] Univ Montreal, Fac Med Vet, Ctr Rech Reprod Anim, St Hyacinthe, PQ J2S 7C6 - Canada

• Document type: Journal article
• Source: PLoS One; v. 9, n. 3 MAR 27 2014.
• Web of Science Citations: 13

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. (AU)

• FAPESP's process: 09/00938-3 - Effect of season on oocyte quality and in vitro embryo production in Holstein cows (Bos taurus) of different categories
• Grantee: Pietro Sampaio Baruselli
• Support Opportunities: Regular Research Grants
• FAPESP's process: 10/13384-3 - Effect of introducing embryonic or somatic mitochondria on development and on mitochondrial inheritance in cattle
• Grantee: Flávio Vieira Meirelles
• Support Opportunities: Regular Research Grants
• FAPESP's process: 11/14207-0 - Effect of season on oocyte quality of buffaloes (Bubalus bubalis): analysis of gene expression and mitochondrial DNA copy number
• Grantee: Carolina Habermann Macabelli
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• FAPESP's process: 10/09561-7 - Effect of introducing a large amount of embryonic or somatic mitochondria on development and on mitochondrial inheritance in bovines
• Grantee: Marcos Roberto Chiaratti
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• FAPESP's process: 12/07510-1 - Molecular evaluation of oocyte maturation and use of cytoplasmic transfer to increse oocyte competence of repeat breeder dairy cows
• Grantee: Roberta Machado Ferreira Saran
• Support Opportunities: Scholarships in Brazil - Post-Doctoral