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, Annexe 4 : Purification de l'ARN total à partir des cellules animales par la « Spin » technologie
, Lyse des cellules en ajoutant un volume approprié de tampon RLT (350 à 600ul)
, Homogénéiser le lysat en le plaçant dans une colonne « QIAshredder spin column » placée dans un collection tube de 2 ml, puis centrifugation à vitesse maximale (13000 rpm) pendant 2 minutes
, Ajouter un volume d'éthanol à 70% au lysat homogénéisé, bien mélanger par pipetage/refoulement
, Transférer jusqu'à 700 ul de l'échantillon à la colonne « RNeasy spin column » placé dans un collection tube de 2 ml. Centrifuger pendant 15 secondes à > 10000 rpm
, Ajouter 700 ul de tampon RLT à la colonne RNeasy. Centrifuger pendant 15 secondes à 10000 rpm pour laver la membrane de la colonne
, Ajouter 500ul de tampon RPE à la colonne RNeasy et centrifuger pendant 2 minutes à 10000 rpm. Cette longue centrifugation va faire sécher la membrane de la colonne pour s'assurer de l'absence de l'éthanol lors de l
, Elution de l'ARN : Placer la colonne RNeasy dans un nouveau tube eppendorf de 1,5 ml. Ajouter 30 à 50 ul d'eau RNAse free directement au centre de la colonne, Centrifuger pendant 1 minute à 10000 rpm