Hostname: page-component-78c5997874-ndw9j Total loading time: 0 Render date: 2024-11-14T13:20:07.973Z Has data issue: false hasContentIssue false
  • English
  • Français

Stability of reference genes for normalization of reverse transcription quantitative real-time PCR (RT-qPCR) data in bovine blastocysts produced by IVF, ICSI and SCNT

Published online by Cambridge University Press:  26 April 2013

Charlotte Luchsinger ,
María Elena Arias ,
Tamara Vargas ,
Marcos Paredes ,
Raúl Sánchez  and
Ricardo Felmer
Charlotte Luchsinger
Affiliation:
Laboratorio de Reproducción, Centro de Biotecnología de La Reproducción (CEBIOR), Facultad de Medicina, Universidad de La Frontera, Temuco, Chile.
María Elena Arias
Affiliation:
Laboratorio de Reproducción, Centro de Biotecnología de La Reproducción (CEBIOR), Facultad de Medicina, Universidad de La Frontera, Temuco, Chile.
Tamara Vargas
Affiliation:
Laboratorio de Reproducción, Centro de Biotecnología de La Reproducción (CEBIOR), Facultad de Medicina, Universidad de La Frontera, Temuco, Chile.
Marcos Paredes
Affiliation:
Laboratorio de Investigación en Biotecnología Animal (LINBA), Facultad de Medicina, Universidad de La Frontera, Temuco, Chile.
Raúl Sánchez
Affiliation:
Laboratorio de Reproducción, Centro de Biotecnología de La Reproducción (CEBIOR), Facultad de Medicina, Universidad de La Frontera, Temuco, Chile.
Ricardo Felmer*
Affiliation:
Laboratorio de Reproducción, Centro de Biotecnología de La Reproducción (CEBIOR), Facultad de Ciencias Agropecuarias y Forestales, Universidad de La Frontera, PO Box 58-D, Temuco, Chile. Departamento de Ciencias Agronómicas y Recursos Naturales, Facultad de Ciencias Agropecuarias y Forestales, Universidad de La Frontera, Temuco, Chile.
*
All correspondence to: Ricardo Felmer. Laboratorio de Reproducción, Centro de Biotecnología de La Reproducción (CEBIOR), Facultad de Ciencias Agropecuarias y Forestales, Universidad de La Frontera, PO Box 58-D, Temuco, Chile. Tel: +56 45 325591. Fax: +56 45 325600. e-mail: [email protected]
Get access Rights & Permissions [Opens in a new window]

Summary

Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is a sensitive and accurate tool for quantitative estimation of gene transcription levels in preimplantation embryos. To control for possible experimental variations, gene expression data must be normalized using internal control genes commonly known as reference genes. However, the stability of reference genes can vary depending on the state of development and/or experimental conditions; hence the assessment of their stability is essential before initiating a gene expression analysis. In the present study, we used RT-qPCR to measure the transcript levels of 10 commonly used reference genes and analyzed their expression stability in bovine blastocysts produced by in vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI) and somatic cell nuclear transfer (SCNT). Using the geNorm program, we found the best combination of genes to normalize gene expression data in bovine embryos at the blastocyst stage produced by IVF (HMBS, SF3A1, and HPRT1), ICSI (H2A, HMBS, and GAPDH), SCNT (ACTB, SF3A1, and SDHA) and/or between blastocysts produced by these methods (GAPDH, HMBS and EEF1A2). We also demonstrated that not only the culture conditions may affect the expression patterns in bovine blastocysts but also the choice of embryo production method may have an important effect.

Keywords

Gene expressionICSIIVFReference genesSCNT
Type
Research Article
Information
Zygote , Volume 22 , Issue 4 , November 2014 , pp. 505 - 512
Copyright
Copyright © Cambridge University Press 2013 

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.)

References

Arias, M.E., Sáchez, R., Risopatrón, J. & Felmer, R.N. (2012). Efecto del pre-tratamiento de espermatozoides bovinos sobre la eficiencia de la inyección intracitoplasmática de espermatozoides (ICSI). Rev. Int. Androl. 10, 18.Google Scholar
Bar, M., Bar, D. & Lehmann, B. (2009). Selection and validation of candidate housekeeping genes for studies of human keratinocytes—review and recommendations. J. Invest. Dermatol. 129, 535–7.CrossRefGoogle ScholarPubMed
Bermejo-Alvarez, P., Rizos, D., Rath, D., Lonergan, P. & Gutierrez-Adan, A. (2008). Epigenetic differences between male and female bovine blastocysts produced in vitro. Physiol. Genomics 32, 264–272.CrossRefGoogle ScholarPubMed
Beyhan, Z., Forsberg, E.J., Eilertsen, K.J., Kent-First, M. & First, N.L. (2007). Gene expression in bovine nuclear transfer embryos in relation to donor cell efficiency in producing live offspring. Mol. Reprod. Dev. 74, 18–27.CrossRefGoogle ScholarPubMed
Bower, N.I., Moser, R.J., Hill, J.R. & Lehnert, S.A. (2007). Universal reference method for real-time PCR gene expression analysis of preimplantation embryos. Biotechniques 42, 199–206.CrossRefGoogle ScholarPubMed
Bustin, S. (2000). Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. J. Mol. Endocrinol. 25, 169–93.CrossRefGoogle ScholarPubMed
Bustin, S. (2002). Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): trends and problems. J. Mol. Endocrinol. 29, 23–39.CrossRefGoogle ScholarPubMed
Bustin, S.A., Benes, V., Garson, J.A., Hellemans, J., Huggett, J., Kubista, M., Mueller, R., Nolan, T., Pfaffl, M.W., Shipley, G.L., Vandesompele, J. & Wittwer, C.T. (2009). The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin. Chem. 55, 611–622.CrossRefGoogle ScholarPubMed
Dheda, K., Huggett, J.F., Bustin, S.A., Johnson, M.A., Rook, G. & Zumla, A. (2004). Validation of housekeeping genes for normalizing RNA expression in real-time PCR. Biotechniques 37, 112–114, 116, 118–119.CrossRefGoogle ScholarPubMed
Felmer, R.N. & Arias, M.E. (2011). Developmental rates of bovine nuclear transfer embryos derived from different fetal non transfected and transfected cells. Electron. J. Biotechnol. 14, 5.CrossRefGoogle Scholar
Felmer, R.N., Arias, M.E., Muñoz, G.A. & Rio, J.H. (2011). Effect of different sequential and two-step culture systems on the development, quality, and RNA expression profile of bovine blastocysts produced in vitro. Mol. Reprod. Dev. 78, 403–14.CrossRefGoogle ScholarPubMed
Goossens, K., Van Poucke, M., Van Soom, A., Vandesompele, J., Van Zeveren, A. & Peelman, L. (2005). Selection of reference genes for quantitative real-time PCR in bovine preimplantation embryos. BMC Dev. Biol. 5, 27.CrossRefGoogle ScholarPubMed
Haller, F., Kulle, B., Schwager, S., Gunawan, B., Heydebreck, A.V., Sültmann, H. & Füzesi, L. (2004). Equivalence test in quantitative reverse transcription polymerase chain reaction: confirmation of reference genes suitable for normalization. Anal. Biochem. 335, 1–9.CrossRefGoogle ScholarPubMed
Huggett, J., Dheda, K., Bustin, S. & Zumla, A. (2005). Real-time RT-PCR normalisation; strategies and considerations. Genes Immun. 6, 279–84.CrossRefGoogle ScholarPubMed
Jemiolo, B. & Trappe, S. (2004). Single muscle fiber gene expression in human skeletal muscle: validation of internal control with exercise. Biochem. Biophys. Res. Commun. 320, 1043–50.CrossRefGoogle ScholarPubMed
Khurana, N.K. & Niemann, H. (2000). Effects of oocyte quality, oxygen tension, embryo density, cumulus cells and energy substrates on cleavage and morula/blastocyst formation of bovine embryos. Theriogenology 54, 741–56.CrossRefGoogle ScholarPubMed
Lazzari, G., Wrenzycki, C., Herrmann, D., Duchi, R., Kruip, T., Niemann, H. & Galli, C. (2002). Cellular and molecular deviations in bovine in vitro-produced embryos are related to the large offspring syndrome. Biol. Reprod. 67, 767–75.CrossRefGoogle Scholar
Livak, K.J. & Schmittgen, T.D. (2001). Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods 25, 402–8.CrossRefGoogle Scholar
Lonergan, P., O'Kearney-Flynn, M. & Boland, M.P. (1999). Effect of protein supplementation and presence of an antioxidant on the development of bovine zygotes in synthetic oviduct fluid medium under high or low oxygen tension. Theriogenology 51, 1565–76.CrossRefGoogle ScholarPubMed
McCurley, A. & Callard, G. (2008). Characterization of housekeeping genes in zebrafish: male-female differences and effects of tissue type, developmental stage and chemical treatment. BMC Mol. Biol. 9, 102.CrossRefGoogle ScholarPubMed
Niemann, H. & Wrenzycki, C. (2000). Alterations of expression of developmentally important genes in preimplantation bovine embryos by in vitro culture conditions: Implications for subsequent development. Theriogenology 53, 21–34.CrossRefGoogle ScholarPubMed
Ohl, F., Jung, M., Xu, C., Stephan, C., Rabien, A., Burkhardt, M., Nitsche, A., Kristiansen, G., Loening, S.A., Radonic, A. & Jung, K. (2005). Gene expression studies in prostate cancer tissue: which reference gene should be selected for normalization? J. Mol. Med. (Berl) 83, 1014–24.CrossRefGoogle ScholarPubMed
Perez, R., Tupac-Yupanqui, I. & Dunner, S. (2008). Evaluation of suitable reference genes for gene expression studies in bovine muscular tissue. BMC Mol. Biol. 9, 79.CrossRefGoogle ScholarPubMed
Pfaffl, M.W. (2001). A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res. 29, e45.CrossRefGoogle ScholarPubMed
Remans, T., Smeets, K., Opdenakker, K., Mathijsen, D., Vangronsveld, J. & 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
Rinaudo, P. & Schultz, R.M. (2004). Effects of embryo culture on global pattern of gene expression in preimplantation mouse embryos. Reproduction 128, 301–11.CrossRefGoogle ScholarPubMed
Rizos, D., Lonergan, P., Boland, M.P., Arroyo-Garcia, R., Pintado, B., de la Fuente, J. & Gutierrez-Adan, A. (2002). Analysis of differential messenger RNA expression between bovine blastocysts produced in different culture systems: implications for blastocyst quality. Biol. Reprod. 66, 589–95.CrossRefGoogle ScholarPubMed
Ross, P.J., Wang, K., Kocabas, A. & Cibelli, J.B. (2010). Housekeeping gene transcript abundance in bovine fertilized and cloned embryos. Cell Reprogram. 12, 709–17.CrossRefGoogle ScholarPubMed
Somers, J., Smith, C., Donnison, M., Wells, D.N., Henderson, H., McLeay, L. & Pfeffer, P.L. (2006). Gene expression profiling of individual bovine nuclear transfer blastocysts. Reproduction 131, 1073–84.CrossRefGoogle ScholarPubMed
Steuerwald, N., Cohen, J., Herrera, R.A. & Brenner, C. (1999). Analysis of gene expression in single oocytes and embryos by real-time rapid cycle fluorescence monitored RT–PCR. Mol. Hum. Reprod. 5, 1034–9.CrossRefGoogle ScholarPubMed
Suzuki, T., Kondo, S., Wakayama, T., Cizdziel, P.E. & Hayashizaki, Y. (2008). Genome-wide analysis of abnormal H3K9 acetylation in cloned mice. PLoS One 3, e1905.CrossRefGoogle ScholarPubMed
Thellin, O., Zorzi, W., Lakaye, B., De Borman, B., Coumans, B., Hennen, G., Grisar, T., Igout, A. & Heinen, E. (1999). Housekeeping genes as internal standards: use and limits. J. Biotechnol. 75, 291–5.CrossRefGoogle Scholar
Vandesompele, 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.CrossRefGoogle ScholarPubMed
Van Guilder, H.D., Vrana, K.E. & Freeman, W.M. (2008). Twenty-five years of quantitative PCR for gene expression analysis. Biotechniques 44, 619–26.CrossRefGoogle ScholarPubMed
Vireque, A.A., Camargo, L.S.A., Serapião, R.V., Rosa e Silva, A.A.M., Watanabe, Y.F., Ferreira, E.M., Navarro, P.A.A.S., Martins, W.P. & Ferriani, R.A. (2009). Preimplantation development and expression of Hsp-70 and Bax genes in bovine blastocysts derived from oocytes matured in alpha-MEM supplemented with growth factors and synthetic macromolecules. Theriogenology 71, 620–7.CrossRefGoogle ScholarPubMed
Walker, C.G., Meier, S., Mitchell, M.D., Roche, J.R. & Littlejohn, M. (2009a). Evaluation of real-time PCR endogenous control genes for analysis of gene expression in bovine endometrium. BMC Mol. Biol. 10, 1–12.CrossRefGoogle ScholarPubMed
Walker, A.M., Kimura, K. & Roberts, M. (2009b). Expression of bovine interferon-tau variants according to sex and age of conceptuses. Theriogenology 72, 44–53.CrossRefGoogle ScholarPubMed
Warrington, J.A., Nair, A., Mahadevappa, M. & Tsyganskaya, M. (2000). Comparison of human adult and fetal expression and identification of 535 housekeeping/maintenance genes. Physiol. Genomics 2, 143–7.CrossRefGoogle ScholarPubMed
Wong, M.L. & Medrano, J.F. (2005). Real-time PCR for mRNA quantitation. Biotechniques 39, 75–85.CrossRefGoogle ScholarPubMed
Zhang, X., Ding, L. & Sandford, A. (2005). Selection of reference genes for gene expression studies in human neutrophils by real-time PCR. BMC Mol. Biol. 6, 4.CrossRefGoogle ScholarPubMed
Zhou, W., Xiang, T., Walker, S., Farrar, V., Hwang, E., Findeisen, B., Sadeghieh, S., Arenivas, F., Abruzzese, R. & Polejaeva, I. (2008). Global gene expression analysis of bovine blastocysts produced by multiple methods. Mol. Reprod. Dev. 75, 744–758.CrossRefGoogle ScholarPubMed