TP - Extraction d'ARN

TP – EXTRACTION D’ARN

Introduction

L’ARN est une molécule monobrin constituée d’assemblage de ribonucléotides. Afin d’étudier cette molécule, il a fallu procéder à son extraction en utilisant le couple Phénol-Chloroforme. Ce dernier permet d’isoler l’ARN des cellules en le séparant des protéines et de l’ADN. L’extraction d’ARN nécessite d’être méticuleux et précautionneux car c’est une molécule extrêmement fragile.

Le but de notre TP est d’extraire l’ARN total ; c'est-à-dire, l’ARN messager, l’ARN de transfert et l’ARN ribosomique d'un tissu cérébral de rat afin de pouvoir quantifier et analyser la qualité de cet ARN par spectrophotométrie.

Matériel et méthodes

Before starting, the centrifuge had to be on, at 4°C. During all the experiments gloves had to be wear. One point five millimeter of ethanol solution at 75% in RNase-free water was prepared. For that 1 125ml of ethanol was mixed to 375µl of water.

Then the tissue preparation started. It had to be in the RNase free tube, and put in the ice tray. RNA is unstable so it needs to be in ice in order to stop the degradation. Five hundred microliter of tri-reagent was added to the tissue and with a piston it was grind and homogenate with the vortex, after that another 500µl of tri-reagent was added. The preparation had a red color, it was centrifuge. These steps permeated to crush the cells and free the RNA which was in the cells’ nucleus; the RNA had to be in the ice as much time as possible.

The RNA had to be separated from the other cells’ component. For that the sample was left at ambient temperature during 5min, and then 200µl of chloroform was added. It was mixed with the vortex and left again at ambient temperature for 10min. After being mixed the preparation had a milky pink color, two phases began to form: a red one at the top and a transparent one at the bottom of the tube.

The preparation was centrifuge during 15mins, at 4°C 13000rpm. The chloroform is denser than the RNA, it goes straight to the bottom and interacts with proteins and DNA, after the preparation was centrifuge it was separated in 3 phases, from the bottom to the top:

Chloroform + DNA and proteins

White interphase

Water-based phase with RNA in it

The RNA was separated from the chloroform, DNA and proteins.

The water-based phase was removed (the ARN) with a pipette and put in another tube. Six hundred microliters were collected, during this step the tube had to be in the same inclination as in the centrifuge. Five hundred microliter of isopropanol was added; the tube was mixed by inverting 5 times, and left at room temperature for 5mins. It was centrifuge during 10min, at 4°C 13000rpm. All the RNA precipitated, afterward the supernatant was removed and the ethanol preparation was added. The tube was slowly stirred in order to wash the RNA and then centrifuge during 5min, at 4°C 13000rpm. The supernatant was thrown without losing the RNA pellet.

The pellet was dried during 5 to 10mins and dissolve in 50µl of RNase-Free water.

Finally 10µl of RNA was diluted in 90µL of RNase-free water. This preparation was used to realize the quantification with a spectrophotometer.

Résultats

Suite à nos manipulations, nous avons examiné la qualité de notre échantillon d’ARN par spectrophotométrie selon le degré de dilution :

Dilution 1/10ème :

〖à A〗_260 :

- Absorbance = 3,068

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