Analyse des gènes

Compte rendu – Travaux Pratiques en méthodes d'investigation en génie biologique

EXTRACTION D'ARN

L'extraction d'ARN est un procédé fréquemment utilisé en biologie moléculaire, dans le but d'étdier l'information génétique quant à la production de protéines et leurs fonctions cellulaires.

Le principe utilisé dans cette manipulation est nommé extraction d’ARN totaux par méthode chimique. Ceci repose sur les propriétés chimiques des molécules qui seront utiles lors des séparations de phase, ce qui va nous permettre de quantifier et d'évaluer la pureté de notre échantillon.

MATERIALS AND METHODS

Before starting work, the centrifuge was tuned at 4°C. It had been necessary to wear gloves,  1,5 mL of 75% ethanol were prepared in Rnase-free : 1,125 mL of ethanol and 0,375 mL of water were sampled and mixed together.  Finally, a piece of ice was taken.

1. 1. Tissue preparation : The tissue sample was put in a Rnase-free tube and the tube with the tissue was put in the ice. Five hundred microliter of TRI-REAGENT were added in the tube. It was homogenized with blue piston pellet in the ice and it was vortexed.. Five hundred microliter of TRI-REAGENT were added and the previous handling with the blue piston pellet was repeated once again.

1. 2. Phase separation : The sample was incubated for 5 minutes at room temperature. Two hundred microliter of chloroform, the tube was closed and the sample was vortexed vigorously for 15 seconds. The tube was incubated at room temperature for 10 minutes. The sample was centrifuged at 13,000 rpm for 15 minutes at 4°C. As the result, the mixture had been separated into red phase, with phenol chloroform, DNA and proteins, a white interphase, and an aqueous phase with RNA.

3. RNA precipitation : Only the aqueous phase was collected and it was put in a new Rnase-free tube. Five hundred microliter of isopropanol were added in the tube. It was mixed five times and incubated for 5 minutes at room temperature. It was centrifuged at 13,000 rpm for 10 minutes at 4°C.

4. RNA wash : The supernatant was removed completely. One microliter of 75% ethanol was added in H2O-RNase free. To wash the RNA, it was mixed and centrifuged at 13,000 rpm for 5 minutes at 4°C. The supernatant was removed without losing RNA.

5. Redissolving RNA : RNA was air-dried for 5 at 10 minutes. The RNA pellet was not let dry completely as this will decrease its solubility. The pellet was dissolved in 50 μL of H2O-RNase free.

1. 6. RNA quantification and control : A dilution was made with a part of extracted RNA at 1:10 in a final volume of 100 μL. Quantification was performed by reading the optical density with a spectrophotometer.

 RESULTATS

Durant l’étape de séparation de phase, après l’ajout du chloroforme, nous avons pu observer que celui-ci est allé immédiatement au fond du tube.

Après avoir ajouté l'isopropanol, nous avons pu également remarquer la formation d'un précipité blanc qui s'atténue après le mélange du tube.

A la fin des manipulation, nous avons fait une dilution au 1/10ème à partir de l'ARN extrait, dans un volume final de 100 µL. Pour ce faire, nous avons donc mélangé 10 µL d'ARN pour 90 µL d'eau.

Ainsi, à l'aide d'un spectrophotomètre réglé sur une longueur d'onde de 260 nm, nous avons déterminé la densité optique (DO) de notre échantillon dilué au 1/10ème. L'absorbance obtenue est de 2,193. Notre facteur de conversion étant 1 unité de DO à 260 nm = 40 µg/mL d'ARN, la concentration d'ARN dilué au 1/10ème était de 87,70091 µg/mL

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