Polysomes immunoprecipitation and isolation of translating RNA from specific cell types of mouse cerebellum - Bertin Instruments

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    Polysomes immunoprecipitation and isolation of translating RNA from specific cell types of mouse cerebellum

    Sources: "Institut de Génomique Fonctionnelle de Lyon (IGFL)", "Ecole Normale Supérieure de Lyon (ENSL)", INRA USC 1370, France

    Context

    Precellys®24 homogenizer has been tested to perform polysomes immunoprecipitation (IP) and translating RNA extraction from IP. In our system, polysomes of some specific cell types of the cerebellum are immunoprecipitated, by expressing a GFP tagged ribosomal protein in these specific cell types with CRE mediated recombination. As these cell types represent a few percent of the whole cerebellum, we should observe an enrichment of specific genes in the IP fraction compared to the input fraction (whole cerebellum lysate before IP).

    Results

    In the IP fraction, we could obtain an enrichment of RNA from our specific cell types (i.e. Purkinje cells and GABAergic interneurons), compared to RNA obtained from whole cerebellum (RNA from input fraction). Genes only expressed by Purkinje cells (Pcp2, Calb1, Fgf7) or genes expressed by both Purkinje cells and GABAergic interneurons (Parv, Gad65) are enriched in the IP fraction, whereas genes that are not expressed in these cell types (Gfap, Mbp, Glast, NeuroD1) are depleted in the IP fraction (Figure 1).

     

    qPCR results obtained on adult mice. Relative normalized expression of different genes in IP fraction (IP23) compared to input fraction (Inp23).

    qPCR results obtained on adult mice. Relative normalized expression of different genes in IP fraction (IP23) compared to input fraction (Inp23).

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