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Validation of reference genes for ovarian tissue from capuchin monkeys (Cebus apella)

Published online by Cambridge University Press:  05 April 2012

A.B. Brito ,
J.S. Lima ,
D.C. Brito ,
L.N. Santana ,
N.N. Costa ,
M.S. Miranda ,
O.M. Ohashi ,
R.R. Santos  and
S.F.S. Domingues
A.B. Brito
Affiliation:
Laboratory of Wild Animal Biology and Medicine, Universidade Federal do Pará, Brazil. Animal Science Post-graduation Program, Universidade Federal do Pará, Brazil.
J.S. Lima
Affiliation:
Laboratory of Wild Animal Biology and Medicine, Universidade Federal do Pará, Brazil. Animal Science Post-graduation Program, Universidade Federal do Pará, Brazil.
D.C. Brito
Affiliation:
Laboratory of Wild Animal Biology and Medicine, Universidade Federal do Pará, Brazil.
L.N. Santana
Affiliation:
Laboratory of Wild Animal Biology and Medicine, Universidade Federal do Pará, Brazil. Animal Science Post-graduation Program, Universidade Federal do Pará, Brazil.
N.N. Costa
Affiliation:
Institute of Biological Sciences, Universidade Federal do Pará, Brazil.
M.S. Miranda
Affiliation:
Institute of Biological Sciences, Universidade Federal do Pará, Brazil.
O.M. Ohashi
Affiliation:
Institute of Biological Sciences, Universidade Federal do Pará, Brazil.
R.R. Santos*
Affiliation:
Rua Augusto Corrêa, Campus Básico, CEP 66075–110, Belém, Pará, Brazil. Laboratory of Wild Animal Biology and Medicine, Universidade Federal do Pará, Brazil. Animal Science Post-graduation Program, Universidade Federal do Pará, Brazil. Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands.
S.F.S. Domingues
Affiliation:
Laboratory of Wild Animal Biology and Medicine, Universidade Federal do Pará, Brazil. Animal Science Post-graduation Program, Universidade Federal do Pará, Brazil.
*
All correspondence to: Regiane R dos Santos. Rua Augusto Corrêa, Campus Básico, CEP 66075–110, Belém, Pará, Brazil. Tel:/Fax: +55 81 32018011. e-mail: r.rodriguesdossantos@pq.cnpq.br
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Summary

There is no tradition in studies reporting the effect of exposure to cryoprotectants or simply hypoxia and hypothermia on gene expression in the ovarian tissue and there has been only one study on reference or target genes quantification, and comparisons of normoxic with hypoxic, hypothermic and toxic conditions. Our aim in the present study was to investigate the stability of three reference genes in the ovarian tissue of capuchin monkeys (Cebus apella). To this end, fresh and cryoprotectant-exposed ovarian biopsies were used. Both fresh and exposed ovarian tissues were subjected to total RNA extraction and synthesis of cDNA. cDNA was amplified by real-time polymerase chain reaction (PCR), and GeNorm, BestKeeper and NormFinder software were used to evaluate the stability of glyceraldehyde-2-phosphate dehydrogenase (GAPDH), hypoxanthine phosphoribosyltransferase 1 (HPRT1) and TATA-binding protein (TBP). Results demonstrated that, in the ovarian tissue from capuchin monkeys, HPRT1 and TBP were the most suitable reference genes and thus could be used as parameters to normalize data in future studies. In contrast, GAPDH appeared as the least stable gene among the tested reference genes. In conclusion, HPRT1 and TBP were the most stable reference genes in fresh and cryoprotectant-exposed ovarian tissue from capuchin monkeys.

Keywords

GAPDHHPRT1Ovarian tissueqRT-PCRStably expressed genesTBP
Type
Research Article
Information
Zygote , Volume 21 , Issue 2 , May 2013 , pp. 167 - 171
Copyright
Copyright © Cambridge University Press 2012

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References

Andersen, C.L., Jensen, J.L. & Orntoft, T.F. (2004). Normalization of real-time quantitative reverse transcription PCR data: a model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets. Cancer Res. 64, 5245–50.CrossRefGoogle Scholar
Domingues, S.F., Caldas-Bussiere, M.C., Martins, N.D & Carvalho, R.A. (2007). Ultrasonographic imaging of the reproductive tract and surgical recovery of oocytes in Cebus apella (capuchin monkeys). Theriogenology 68, 1251–9.CrossRefGoogle ScholarPubMed
Fink, T., Lund, P., Pilgaard, L., Rasmussen, J.G., Duroux, M. & Zachar, V. (2008). Instability of standard PCR reference genes in adipose-derived stem cells during propagation, differentiation and hypoxic exposure. BMC Mol. Biol. 9, 98.CrossRefGoogle ScholarPubMed
Foldager, C.B., Munir, S., Ulrik-Vinther, W., Soballe, K., Bunger, C. & Lind, M. (2009). Validation of suitable house keeping genes for hypoxia-culture human chondrocytes. BMC Mol. Biol. 10, 94.CrossRefGoogle Scholar
Frota, I.M., Leitão, C.C., Costa, J.J., Brito, I.R., Van den Hurk, R. & Silva, J.R. (2011). Stability of housekeeping genes and expression of locally produced growth factors and hormone receptors in goat preantral follicles. Zygote 19, 71–83.CrossRefGoogle ScholarPubMed
Gubern, C., Hurtado, O., Rodriguez, R., Morales, J.R., Romera, V.G., Moro, M.A., Lizasoain, I., Serena, J. & Mallolas, J. (2009). Validation of housekeeping genes for quantitative real-time PCR in in-vivo and in-vitro models of cerebral ischaemia. BMC Mol. Biol. 10: 57.CrossRefGoogle ScholarPubMed
Hugget, J., Dheda, K., Bustin, S. & Zumla, A. (2005). Real-time RT-PCR normalization; strategies and considerations. Genes Immun. 6: 279–284.CrossRefGoogle Scholar
Isachenko, V., Lapidus, I., Isachenko, E., Krivokharchenko, A., Kreienberg, R., Woriedh, M., Bader, M. & Weiss, J.M. (2009). Human ovarian tissue vitrification versus conventional freezing: morphological, endocrinological, and molecular biological evaluation. Reproduction 138: 319–27.CrossRefGoogle ScholarPubMed
Kopitz, A., Soppa, J., Kreitschi, C. & Hauser, K. (2009). Differential stability of TATA-box binding proteins from archaea with different optimal growth temperatures. Spectrochim. Acta A. Mol. Biomol. Spectrosc. 73, 799–804.CrossRefGoogle ScholarPubMed
Li, Y.L., Ye, F., Hu, Y., Lu, W.G. & Xie, X. (2009). Identification of suitable reference genes for gene expression studies of human serous ovarian cancer by real-time polymerase chain reaction. Anal. Biochem. 394, 110–6.CrossRefGoogle ScholarPubMed
Lucas, E.S., Watkins, A.J., Cox, A.L., Marfy-Smith, S.J., Smyth, N. & Fleming, T.P. (2011). Tissue-specific selection of reference genes is required for expression studies in the mouse model of maternal protein undernutrition. Theriogenology 76, 558–69.CrossRefGoogle Scholar
Meldgaard, M., Fenger, C., Lambertsen, K.L., Pedersen, M.D., Ladeby, R. & Finsen, B. (2006). Validation of two reference genes for mRNA level studies of murine disease models in neurobiology. J. Neurosci. Methods 156: 101–10.CrossRefGoogle ScholarPubMed
Pfaffl, M.W., Tichopad, A., Prgomet, C. & Neuvians, T.P. (2004). Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper Excel-based tool using pair-wise correlations. Biotechnol. Lett. 26, 509–15.CrossRefGoogle ScholarPubMed
Seoul, D., Choe, H., Zheng, H., Jang, K., Ramakrishnan, P.S., Lim, T.H. & Martin, J.A. (2011). Selection of reference genes for normalization of quantitative real-time PCR in organ culture of the rat and rabbit intervertebral disc. BMC Res. Notes. 26, 162.CrossRefGoogle Scholar
Svingen, T., Spilller, C.M., Kashimada, K., Harley, V.R. & Koopman, P. (2009). Identification of suitable normalizing genes for quantitative real-time RT-PCR analysis of gene expression in fetal mouse gonads. Sex. Dev. 3: 194–204.CrossRefGoogle ScholarPubMed
Vandersompele, J., De Preter, K., Pattyn, F., Poppe, B., Van Roy, N., De Paepe, A. & Speleman, F. (2002). Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol. 3, RESEARCH0034.Google Scholar