Hostname: page-component-cd9895bd7-8ctnn Total loading time: 0 Render date: 2024-12-27T10:20:02.910Z Has data issue: false hasContentIssue false
  • English
  • Français

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
Get access Rights & Permissions [Opens in a new window]

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

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

Footnotes

*

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

References

Bakhtari, A and Ross, PJ (2014). DPPA3 prevents cytosine hydroxymethylation of the maternal pronucleus and is required for normal development in bovine embryos. Epigenetics 9, 1271–9.CrossRefGoogle ScholarPubMed
Bebbere, DBogliolo, LAriu, FFois, SLeoni, GGSuccu, SBerlinguer, F and Ledda, S (2010). Different temporal gene expression patterns for ovine pre-implantation embryos produced by parthenogenesis or in vitro fertilization. Theriogenology 74, 712–23.CrossRefGoogle ScholarPubMed
Bernardi, MLCotinot, CPayen, E and Delouis, C (1996). Transcription of Y- and X-linked genes in preimplantation ovine embryos. Mol Reprod Dev 45, 132–8.3.0.CO;2-U>CrossRefGoogle Scholar
Bustin, SA (2010). Why the need for qPCR publication guidelines? – The case for MIQE. Methods 50, 217–226.CrossRefGoogle ScholarPubMed
Chapman, JR and Waldenström, J (2015). With reference to reference genes: a systematic review of endogenous controls in gene expression studies. PLoS One 10, e0141853.CrossRefGoogle ScholarPubMed
Derveaux, S, Vandesompele, J and Hellemans, J (2010). How to do successful gene expression analysis using real-time PCR. Methods 50, 227–30.CrossRefGoogle Scholar
Goossens, K, Van Poucke, M, Van Soom, A, Vandesompele, J, Van Zeveren, A and Peelman, LJ (2005). Selection of reference genes for quantitative real-time PCR in bovine preimplantation embryos. BMC Dev Biol 5, 27.10.1186/1471-213X-5-27CrossRefGoogle ScholarPubMed
Hosseini, SM, Moulavi, F, Tanhaie-Vash, N, Asgari, V, Ghanaei, HR, Abedi-Dorche, M, Jafarzadeh, N, Gourabi, H, Shahverdi, AH, Dizaj, AV, Shirazi, A and Nasr-Esfahani, MH (2016). The principal forces of oocyte polarity are evolutionary conserved but may not affect the contribution of the first two blastomeres to the blastocyst development in mammals. PLoS One 11, e0148382.CrossRefGoogle Scholar
Jeong, JK, Kang, MH, Gurunathan, S, Cho, SG, Park, C, Seo, HG and Kim, JH (2014). Evaluation of reference genes in mouse preimplantation embryos for gene expression studies using real-time quantitative RT-PCR (RT-qPCR). BMC Res Notes 7, 675.CrossRefGoogle Scholar
Li, L, Zheng, P and Dean, J (2010). Maternal control of early mouse development. Development 137, 859–70.CrossRefGoogle ScholarPubMed
Luchsinger, C, Arias, ME, Vargas, T, Paredes, M, Sánchez, R and Felmer, R (2014). Stability of reference genes for normalization of reverse transcription quantitative real-time PCR (RT-qPCR) data in bovine blastocysts produced using IVF, ICSI and SCNT. Zygote 22, 505–12.CrossRefGoogle ScholarPubMed
Masala, L, Ariu, F, Bogliolo, L, Bellu, E, Ledda, S and Bebbere, D (2018). Delay in maternal transcript degradation in ovine embryos derived from low competence oocytes. Mol Reprod Dev 85, 427–39.CrossRefGoogle ScholarPubMed
Moura, MT, Silva, RL, Cantanhêde, LF, Silva, JB, Ferreira-Silva, JC, Silva, PG, Ramos-Deus, P, Pandolfi, V, Kido, EA, Benko-Iseppon, AM and Oliveira, MAL (2018). Activity of non-canonical pluripotency-associated transcription factors in goat cumulus–oocyte complexes. Livestock Sci 212, 52–6.CrossRefGoogle Scholar
Moura, MT, Silva, RL, Nascimento, PS, Ferreira-Silva, JC, Cantanhêde, LF, Kido, EA, Benko-Iseppon, AM and Oliveira, MAL (2019) Inter-genus gene expression analysis in livestock fibroblasts using reference gene validation based upon a multi-species primer set. PLoS One 14, e0221170.CrossRefGoogle ScholarPubMed
Nakamura, T, Arai, Y, Umehara, H, Masuhara, M, Kimura, T, Taniguchi, H, Sekimoto, T, Ikawa, M, Yoneda, Y, Okabe, M, Tanaka, S, Shiota, K and Nakano, T (2007). PGC7/Stella protects against DNA demethylation in early embryogenesis. Nat Cell Biol 9, 64–71.CrossRefGoogle ScholarPubMed
Nakamura, T, Liu, YJ, Nakashima, H, Umehara, H, Inoue, K, Matoba, S, Tachibana, M, Ogura, A, Shinkai, Y and Nakano, T (2012). PGC7 binds histone H3K9me2 to protect against conversion of 5mC to 5hmC in early embryos. Nature 486, 415–9.CrossRefGoogle ScholarPubMed
O’Connor, T, Wilmut, I and Taylor, J (2013). Quantitative evaluation of reference genes for real-time pcr during in vitro maturation of ovine oocytes. Reprod Domest Anim 48, 477–83.CrossRefGoogle ScholarPubMed
Payer, B, Saitou, M, Barton, SC, Thresher, R, Dixon, JP, Zahn, D, Colledge, WH, Carlton, MB, Nakano, T and Surani, MA (2003). Stella is a maternal effect gene required for normal early development in mice. Curr Biol 13, 2110–7.CrossRefGoogle ScholarPubMed
Pfaffl, MW, Horgan, GW and Dempfle, L (2002). Relative expression software tool (REST) for group-wise comparison and statistical analysis of relative expression results in real-time PCR. Nucleic Acids Res 30, e36.CrossRefGoogle ScholarPubMed
Remans, T, Smeets, K, Opdenakker, K, Mathijsen, D, Vangronsveld, J and Cuypers, A (2008). Normalisation of real-time RT-PCR gene expression measurements in Arabidopsis thaliana exposed to increased metal concentrations. Planta 227, 1343–9.CrossRefGoogle ScholarPubMed
Rozen, S and Skaletsky, H (2000). Primer3 on the WWW for general users and for biologist programmers. Methods Mol Biol 132, 365–86.Google ScholarPubMed
Schmittgen, TD and Livak, KJ (2008). Analyzing real-time PCR data by the comparative C T method. Nat Protocols 3, 1101–8.CrossRefGoogle ScholarPubMed
Silva, RLO, Silva, MD, Neto, F, Costa, JR, Nardi, CHD, Chabregas, SM, Burnquist, WL, Kahl, G, Benko-Iseppon, AM and Kido, EA (2014). Validation of novel reference genes for reverse transcription quantitative real-time PCR in drought-stressed sugarcane. Sci World J 2014, 1–12.Google ScholarPubMed
Silva, PGC, Moura, MT, Silva, RLO, Nascimento, PS, Silva, JB, Ferreira-Silva, JC, Cantanhêde, LF, Chaves, MS, Benko-Iseppon, AM and Oliveira, MAL (2018). Temporal expression of pluripotency-associated transcription factors in sheep and cattle preimplantation embryos. Zygote 26, 270–8.CrossRefGoogle ScholarPubMed
Thélie, A, Papillier, P, Pennetier, S, Perreau, C, Traverso, JM, Uzbekova, S, Mermillod, P, Joly, C, Humblot, P and Dalbiès-Tran, R (2007). Differential regulation of abundance and deadenylation of maternal transcripts during bovine oocyte maturation in vitro and in vivo. BMC Dev. Biol. 7, 125.CrossRefGoogle ScholarPubMed
Thurston, A, Taylor, J, Gardner, J, Sinclair, KD and Young, LE (2008). Monoallelic expression of nine imprinted genes in the sheep embryo occurs after the blastocyst stage. Reproduction 135, 29–40.CrossRefGoogle ScholarPubMed
Vandesompele, J, De Preter, K, Pattyn, F, Poppe, B, Van Roy, N, De Paepe, A and Speleman, F (2002). Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol. 3, research 0034-1.CrossRefGoogle ScholarPubMed
VanGuilder, HD, Vrana, KE and Freeman, WM (2008). Twenty-five years of quantitative PCR for gene expression analysis. Biotechniques 44, 619–26.CrossRefGoogle ScholarPubMed
Wang, YK, Li, X, Song, ZQ and Yang, CX (2017). Methods of RNA preparation affect mRNA abundance quantification of reference genes in pig maturing oocytes. Reprod Domest Anim 52, 722–30.CrossRefGoogle ScholarPubMed
Wennekamp, S and Hiiragi, T (2012). Stochastic processes in the development of pluripotency in vivo. Biotechnol J 7, 737–44.CrossRefGoogle ScholarPubMed
Ye, J, Coulouris, G, Zaretskaya, I, Cutcutache, I, Rozen, S and Madden, TL (2012). Primer-BLAST: a tool to design target-specific primers for polymerase chain reaction. BMC Bioinform 13, 134.CrossRefGoogle ScholarPubMed
Zhao, S, Xu, J, Liu, S, Cui, K, Li, Z and Liu, N (2018). Dppa3 in pluripotency maintenance of ES cells and early embryogenesis. J Cell Biochem 120, 4794–9.CrossRefGoogle ScholarPubMed
Supplementary material: PDF

Nascimento et al. supplementary material

Nascimento et al. supplementary material

Download Nascimento et al. supplementary material(PDF)
PDF 1.8 MB