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(Reference retrieved automatically from SciELO through information on FAPESP grant and its corresponding number as mentioned in the publication by the authors.)

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

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Author(s):
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]
Total Authors: 7
Affiliation:
[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
Total Affiliations: 7
Document type: Journal article
Source: Animal Reproduction; v. 16, n. 2, p. 290-296, 2019-11-25.
Abstract

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)

FAPESP's process: 12/16444-2 - Impact of phospholipid supplementation on culture medium and/or vitrification medium on bovine oocyte quality, cryotolerance and gene expression
Grantee:Caroline Palmieri Pitangui Molina
Support Opportunities: Scholarships in Brazil - Doctorate
FAPESP's process: 12/11069-9 - ANDROGEN RECEPTOR CAG POLIMORFISM AND TELOMERE LENGTH IN PREMATURE OVARIAN FAILURE
Grantee:Cristiana Libardi Miranda Furtado
Support Opportunities: Scholarships in Brazil - Post-Doctoral
FAPESP's process: 12/06006-8 - Gene expression comparison between bovine oocytes matured in vivo or in vitro, and evaluation of the vitrification influence in this expression
Grantee:Lisandra Cristina Caetano da Silva
Support Opportunities: Scholarships in Brazil - Post-Doctoral
FAPESP's process: 12/06068-3 - Gene expression comparison between bovine oocytes matured in vivo or in vitro, and evaluation of the vitrification influence in this expression
Grantee:Ana Carolina Japur de Sá Rosa e Silva
Support Opportunities: Regular Research Grants