Methods for isolating and characterizing endogenous...

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G01N 33/53 (2006.01) C12Q 1/68 (2006.01) G01N 33/543 (2006.01) G01N 33/68 (2006.01)

Patent

CA 2396058

Cellular mRNA-protein (mRNP) complexes are partitioned in vivo by contacting a biological sample with at least one ligand that specifically binds at least one component of a mRNP complex. Suitable biological samples comprise at least one mRNA-protein (mRNP) complex and include cell cultures, cell extracts, and whole tissue, including tumor tissue. Ligands include antibodies that specifically bind RNA-binding or RNA-associated proteins present in the mRNP complex. The mRNP complex is separated by binding the ligand with a binding molecule specific for the ligand, where the binding molecule is attached to a solid support. The mRNP complex is collected by removing the mRNP complex from the solid support. After collecting the mRNP complex, the mRNA bound within the complex may be characterized and identified. Subsets of the total mRNA population of a cell may accordingly be characterized, and a gene expression profile of the cell obtained.

On divise in vivo des complexes constitués par ARNm cellulaire et des protéines (RNPm) par mise en contact d'un spécimen biologique avec au moins un ligand se liant de façon spécifique à au moins un constituant du complexe RNPm. Des spécimens biologiques appropriés consistent en un complexe ARNm-protéine (RNPm) et comprennent des cultures cellulaires, des extraits cellulaires et des tissus entiers, y compris des tissus tumoraux. Ces ligands consistent en des anticorps se liant de façon spécifique à des protéines de fixation ARN ou associées à ARN présentes dans le complexe RNPm. On sépare ce dernier par fixation du ligand à une molécule de fixation spécifique pour ce ligand, la molécule de fixation étant fixée à un support solide. On recueille ce complexe RNPm par suppression dudit complexe du support solide. Après avoir recueilli ce complexe RNPm, on peut caractériser et identifier l'ARNm ayant fait l'objet d'une liaison à l'intérieur de ce complexe. On peut, en conséquence, caractériser des sous-ensembles de la totalité de la population d'ARNm d'une cellule et obtenir le profil d'expression génétique de cette cellule.

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