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Housekeeping genes for RT-qPCR in ovine preimplantation embryos

Published online by Cambridge University Press:  30 July 2020

Pábola Santos Nascimento ,
Marcelo Tigre Moura Open the ORCID record for Marcelo Tigre Moura [Opens in a new window] ,
Roberta Lane Oliveira Silva ,
Pamela Ramos-Deus ,
José Carlos Ferreira-Silva ,
Joane Isis Travassos Veira ,
Antônio Santana Santos Filho ,
Sebastião Inocêncio Guido ,
Cláudio Coutinho Bartolomeu  and
Ana Maria Benko-Iseppon
...Show all authors
Pábola Santos Nascimento
Affiliation:
Departamento de Medicina Veterinária, Universidade Federal Rural de Pernambuco, Brazil
Marcelo Tigre Moura*
Affiliation:
Departamento de Medicina Veterinária, Universidade Federal Rural de Pernambuco, Brazil
Roberta Lane Oliveira Silva
Affiliation:
Departamento de Genética, Universidade Federal de Pernambuco, Brazil
Pamela Ramos-Deus
Affiliation:
Departamento de Medicina Veterinária, Universidade Federal Rural de Pernambuco, Brazil
José Carlos Ferreira-Silva
Affiliation:
Departamento de Medicina Veterinária, Universidade Federal Rural de Pernambuco, Brazil
Joane Isis Travassos Veira
Affiliation:
Departamento de Medicina Veterinária, Universidade Federal Rural de Pernambuco, Brazil
Antônio Santana Santos Filho
Affiliation:
Instituto Agronômico de Pernambuco, Arcoverde, Pernambuco, Brazil
Sebastião Inocêncio Guido
Affiliation:
Instituto Agronômico de Pernambuco, Arcoverde, Pernambuco, Brazil
Cláudio Coutinho Bartolomeu
Affiliation:
Departamento de Medicina Veterinária, Universidade Federal Rural de Pernambuco, Brazil
Ana Maria Benko-Iseppon
Affiliation:
Departamento de Genética, Universidade Federal de Pernambuco, Brazil
Marcos Antonio Lemos Oliveira
Affiliation:
Departamento de Medicina Veterinária, Universidade Federal Rural de Pernambuco, Brazil
*
Author for correspondence: Marcelo Tigre Moura. Laboratório de Biotécnicas Aplicadas à Reprodução, Departamento de Medicina Veterinária, Universidade Federal Rural de Pernambuco, Av. Dom Manoel de Medeiros s/n, Dois Irmãos, Recife, CEP. 52171-900. Brazil. Tel: +55 8133206414. E-mail: marcelotmoura@gmail.com
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Summary

Housekeeping genes (HKG) are paramount for accurate gene expression analysis during preimplantation development. Markedly, quantitative reverse transcription polymerase chain reaction (RT-qPCR) in ovine embryos currently lacks HKGs. Therefore, we tested 11 HKGs for RT-qPCR normalization during ovine parthenogenetic preimplantation development. Seven HKGs reached the qPCR efficiency threshold (97.20–105.96%), with correlation coefficients ranging from −0.922 to −0.998 and slopes from −3.22 to −3.59. GeNorm ranked glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and TATA-binding protein (TBP) as the best HKG pair, while H3 histone, family 3A (H3F3A) was the third HKG. Relative gene expression was measured for zinc finger protein X-linked (ZFX) and developmental pluripotency-associated 3 (DPPA3) transcripts during ovine parthenogenetic preimplantation development. ZFX did not show any transcript abundance fluctuation among oocytes, cleavage-stage embryos, and morulae. DPPA3 transcript abundance was also similar among all developmental stages, therefore suggesting that it may not display a maternal gene expression profile. In silico analysis of ovine DPPA3 mRNA and protein showed high conservation to bovine orthologues. However, DPPA3 orthologues differed in regulatory motifs. In conclusion, GAPDH, TBP and H3F3A are stable HKGs in ovine parthenogenetic embryos and allow accurate RT-qPCR-based gene expression analysis.

Keywords

EmbryogenesisPGC7Real-time PCRReference geneSTELLA
Type
Research Article
Information
Zygote , Volume 28 , Issue 5 , October 2020 , pp. 432 - 439
Copyright
© The Author(s), 2020. Published by Cambridge University Press

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Footnotes

*

Present address: Laboratory of Cell Biology, Federal University of São Paulo – UNIFESP, Diadema - SP, Brazil.

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