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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: rfelmerd@gmail.com
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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 

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