title: Method to isolate polyribosomal mRNA from scarce samples such as mammalian oocytes and early embryos.
abstract: BACKGROUND
  Although the transcriptome of minute quantities of cells can be profiled using nucleic acid amplification techniques, it remains difficult to distinguish between active and stored messenger RNA. Transcript storage occurs at specific stages of gametogenesis and is particularly important in oogenesis as stored maternal mRNA is used to sustain de novo protein synthesis during the early developmental stages until the embryonic genome gets activated. In many cases, stored mRNA can be several times more abundant than mRNA ready for translation. In order to identify active mRNA in bovine oocytes, we sought to develop a method of isolating very small amounts of polyribosome mRNA.
  
  
  RESULTS
  The proposed method is based on mixing the extracted oocyte cytoplasm with a preparation of polyribosomes obtained from a non-homologous source (Drosophila) and using sucrose density gradient ultracentrifugation to separate the polyribosomes. It involves cross-linking the non-homologous polyribosomes and neutralizing the cross-linking agent. Using this method, we show that certain stages of oocyte maturation coincide with changes in the abundance of polyribosomal mRNA but not total RNA or poly(A). We also show that the abundance of selected sequences matched changes in the corresponding protein levels.
  
  
  CONCLUSIONS
  We report here the successful use of a method to profile mRNA present in the polyribosomal fraction obtained from as little as 75 mammalian oocytes. Polyribosomal mRNA fractionation thus provides a new tool for studying gametogenesis and early development with better representation of the underlying physiological status.
authors.affiliation: Laboratoire de génomique fonctionnelle du développement embryonnaire, Centre de recherche en biologie de la reproduction, Pavillon Comtois, Faculté des sciences de l'agriculture et de l'alimentation, Université Laval, Québec, G1V 0A6, Canada. claude.robert@fsaa.ulaval.ca
authors.forename: Sara
authors.initials: S
authors.lastname: Scantland
authors.forename: Jean-Philippe
authors.initials: JP
authors.lastname: Grenon
authors.forename: Marie-Hélène
authors.initials: MH
authors.lastname: Desrochers
authors.forename: Marc-André
authors.initials: MA
authors.lastname: Sirard
authors.forename: Edward W
authors.initials: EW
authors.lastname: Khandjian
authors.forename: Claude
authors.initials: C
authors.lastname: Robert
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002417:Cattle; D002499:Centrifugation, Density Gradient; D004622:Embryo, Mammalian / Q000166:cytology; D009865:Oocytes / Q000166:cytology; D016133:Polymerase Chain Reaction; D011132:Polyribosomes / Q000737:chemistry*; D012333:RNA, Messenger / Q000302:isolation & purification*; D015203:Reproducibility of Results
chemical_list: D012333:RNA, Messenger