Validation of reference genes for gene expression studies in bovine oocytes and cumulus cells derived from in vitro maturation

Autor(es)

Lisandra Cristina Caetano ^[1] ; Cristiana Libardi Miranda-Furtado ^[2] ; Luciene Aparecida Batista ^[3] ; Caroline Palmieri Pitangui-Molina ^[4] ; Thaís Tiemi Higa ^[5] ; Cristiana Carolina Padovan ^[6] ; Ana Carolina Japur de Sá Rosa-e-Silva ^[7]

Afiliação do(s) autor(es)

^[1] University of São Paulo. Ribeirão Preto Medical School. Department of Gynecology and Obstetrics - Brasil

^[2] University of São Paulo. Ribeirão Preto Medical School. Department of Gynecology and Obstetrics - Brasil

^[3] University of São Paulo. Ribeirão Preto Medical School. Department of Gynecology and Obstetrics - Brasil

^[4] University of São Paulo. Ribeirão Preto Medical School. Department of Gynecology and Obstetrics - Brasil

^[5] University of São Paulo. Ribeirão Preto Medical School. Department of Gynecology and Obstetrics - Brasil

^[6] University of São Paulo. Ribeirão Preto Medical School. Department of Gynecology and Obstetrics - Brasil

^[7] University of São Paulo. Ribeirão Preto Medical School. Department of Gynecology and Obstetrics - Brasil

• Tipo de documento: Artigo Científico
• Fonte: Animal Reproduction; v. 16, n. 2, p. 290-296, 2019-11-25.

Resumo

Abstract Quantitative real-time PCR (qPCR) is a valuable tool for gene expression studies and it is necessary to choose an ideal endogenous reference gene for data normalization. This work studied a set of reference genes in oocytes and cumulus cells of COCs (Cumulus-Oocyte Complexes) that are suitable for relative gene expression analyses after in vitro maturation (IVM) in bovine. Immature COCs were collected from ovaries of Nelore cattle (Bos indicus) and submitted to IVM. MII oocytes and cumulus cells were subjected to RNA extraction, reverse transcription and preamplification of cDNA. The expression level of eight reference genes (ACTB, GADPH, B2M, H2AFZ, GUSB, HPRT1, PPIA, and TBP) was measured by real time PCR and analyzed by geNorm software. The gene stability measure (M) was calculated and the ideal number of reference genes (RGs) was determined by the V value (pairwise variation). For oocyte samples, two RGs were the ideal number for relative quantification: HPRT1 and B2M and for bovine cumulus samples four were indicated: HPRT1, PPIA, B2M, and TBP genes. The normalization of a non-reference target gene (SOD1) by these reference genes was shown to be considerably different from normalization by less stable reference genes. Our results strengthen the importance of choosing good normalizing genes in order to analyze gene expression under specific experimental conditions and we suggest the use of these RGs in oocytes and cumulus cells of bovine cattle in in vitro matured COCs. (AU)

• Processo FAPESP: 12/11069-9 - Polimorfismo cag do receptor de andrógenos e comprimento do telômero em pacientes com falência ovariana prematura
• Beneficiário: Cristiana Libardi Miranda Furtado
• Modalidade de apoio: Bolsas no Brasil - Pós-Doutorado
• Processo FAPESP: 12/06006-8 - Comparação da expressão gênica entre oócitos bovinos maturados in vivo e in vitro, e da influência do processo de vitrificação sobre esta expressão.
• Beneficiário: Lisandra Cristina Caetano da Silva
• Modalidade de apoio: Bolsas no Brasil - Pós-Doutorado
• Processo FAPESP: 12/16444-2 - Suplementação do meio de maturação in vitro e vitrificação com fosfolipídio e seu impacto na criotolerância, qualidade oocitária e expressão gênica em oócitos, usando o bovino como modelo experimental.
• Beneficiário: Caroline Palmieri Pitangui Molina
• Modalidade de apoio: Bolsas no Brasil - Doutorado
• Processo FAPESP: 12/06068-3 - Comparação da expressão gênica entre oócitos bovinos maturados in vivo e in vitro, e da influência do processo de vitrificação sobre esta expressão
• Beneficiário: Ana Carolina Japur de Sá Rosa e Silva
• Modalidade de apoio: Auxílio à Pesquisa - Regular