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V. Figure, Schéma du dispositif permettant la préparation d'émulsions. La sonde à ultrasons est plongée au plus profond du tube Falcon? sans toutefois toucher la paroi

. Dans-le-cas-d, une émulsion double, une première émulsion eau dans huile (w/o) est préparée en mélangeant 400 µL de phase aqueuse dite « interne

. Historique-de-l-'électrophorèse-les-travaux-sur-l-'électrophorèse-remontent-au-milieu-du-xix-Ème-siècle and . Au-phénomène-appelé-cataphorèse-développé-par-quincke, Il a montré que l'on peut déplacer des particules colloïdales sous l'action d'un champ électrique. Helmholtz mettra en évidence par son concept d'électro-osmose que les espèces chargées migrent vers l'électrode opposée à leur charge

K. W. Le-chercheur-suédois-arne, Tiselius (prix Nobel de chimie 1948) est considéré comme le père de l'électrophorèse moderne, 1937.

P. Le, a pas pu être testé car il n'est pas dispersable en l'état dans l'eau, il précipite lorsqu'on évapore le solvant organique après formation de l'émulsion

F. Boulogne and . Supplémenté-en-pénicilline-et-en-streptomycine, Il est ensuite soumis à une digestion par une solution de pronase (2 mg/ml) (Sigma Aldrich, USA) dans du NaCl 0,9 % durant deux heures à 37 °C sous 5 % de CO 2 Après trois rinçages par du NaCl 0,9 %, le cartilage est soumis à une deuxième digestion par une solution de collagénase B (1,5 mg/ml) (Roche, Suisse) pendant une nuit à 37 °C sous 5 % de CO 2 . La collagénase B permet la libération des chondrocytes par destructuration de la matrice extra cellulaire, Elle est reconstituée dans du milieu de culture DMEM/F12 (Gibco ® , USA) supplémenté à 1 % en pénicilline et en streptomycine

. Transcriptase-inverse, La réction de transcription inverse se déroule pendant 1 heure à 37 °C. Les ARN restants sont dénaturés par la chaleur (5 minutes à 95°C)

. Dans-un-premier and . Temps, ADNc (ADN complémentaire) sont dénaturés à 95 °C, ce qui entraîne une séparation des deux brins d'ADN, puis les amorces oligonucléotidiques spécifiques vont se fixer sur les brins d'ADN : c'est l'étape d'hybridation

. Enfin, °C, la Taq-polymérase synthétisant le brin complémentaire après la séquence d'hybridation, côté 3'. Ces cycles sont répétés en général une quarantaine de fois ; la quantité d'ADN correspondant au gène, doublant à chaque cycle

. Au-début-de-la-réaction,-le-mélange-réactionnel-contient-de-l-'adn and . Le-fluorophore-non-lié, Après hybridation des amorces, le fluorophore se lie au double brin. La liaison à l'ADN se traduit par une augmentation de la fluorescence. Pendant l'étape d'élongation, le nombre de molécules de fluorophore lié à l'ADN synthétisé augmente ce qui se traduit par une augmentation de la fluorescence. a) Exploitation des données

V. Figure, Quantification de l'expression d'un gène en fonction d'une gamme d'ADNc

. Chaque-extrait-d, ADNc obtenu après la transcription inverse est dilué au quart dans de l'eau ultra pure

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