Method to isolate polyribosomal mRNA from scarce samples such as mammalian oocytes and early embryos
1 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
2 Laboratoire de génomique et protéomique animales, Centre de recherche en biologie de la reproduction, Pavillon INAF, Département des sciences animales, Faculté des sciences de l'agriculture et de l'alimentation, Université Laval, Québec, G1V 0A6, Canada
3 Neurobiologie cellulaire, Centre de recherche Robert-Giffard, Département de psychiatrie et de neurosciences, Faculté de Médecine, Université Laval, Québec, G1V 0A6, Canada
BMC Developmental Biology 2011, 11:8 doi:10.1186/1471-213X-11-8Published: 15 February 2011
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.
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.
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.