Quantification of housekeeping transcript levels during the development of bovine preimplantation embryos

Claude Robert, Serge McGraw, Lyne Massicotte, Marco Pravetoni, Fulvio Gandolfi, Marc André Sirard

Research output: Contribution to journalArticle

177 Scopus citations

Abstract

In mammals, the study of gene expression in the preimplantation embryo has been difficult because the standard procedures used to quantify mRNA generally require large amounts of starting material. The development of protocols using different quantitative strategies generally involving the polymerase chain reaction (PCR) has provided new tools for exploration of gene expression in preimplantation embryos. However, the use of an internal standard, often referred as a housekeeping gene, is essential to normalize the mRNA levels. RNA levels of eight housekeeping genes were quantified using real time PCR throughout the preimplantation period of the bovine embryo to find the most suitable gene to be used as standard. Histone H2a was the best internal standard because the transcript levels were constant across the preimplantation period. Linear amplification of antisense RNA using the T7 promotor for in vitro transcription of the entire RNA pool was evaluated as a suitable way to preamplify the starting material prior to quantification and was effective in providing accurate RNA abundance profiles throughout the preimplantation period. However, the amplification appears to be template dependent because the amplification factors were higher for some genes.

Original languageEnglish (US)
Pages (from-to)1465-1472
Number of pages8
JournalBiology of reproduction
Volume67
Issue number5
DOIs
StatePublished - Nov 1 2002
Externally publishedYes

Keywords

  • Developmental biology
  • Early development
  • Embryo
  • Oocyte development
  • Ovum

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