RNA Isolation Protocol (by Ehab Lab)

Preparation: 1.Clean the bench, pipetes, pipet tip boxes with 50 % belach:ethanol

1. Cool centrifuge 4 C
2. Cold isopropanol and 75 % ethanol
3. RNAase free tubes, filter tips and water should be ready

PROTOCOL:

Homogenizaiton

1. Homogenize tissue samples in TRI Reagent
2. Vortex
3. Freze-throw x3 times for 10 min at -150 C

Phase Seperation

1. Store the homogenate for 5 min at RT to permit the complete dissocation of nucleoprotein complexes.
2. Suplement the homogenate with 0.2 ml chloroform per 1 ml of TRI Reagent
3. Vortex for 15 seconds.
4. Store the resulting mixture at room temperature for 2-15 minutes and centrifuge at full speed g for 30 min at 4 C.
5. Collect the RNA from upper phase to fresh RNAase free tube.

RNA Precipitation 1.Use 0.5 ml ofcold isopropanol per 1 ml of TRI Reagent

1. Store samples 10 min at – 80
2. Wait until they completely dissolve and centrifuge at full speed g for 30 min at 4 C.
3. RNA precipitate (often invisible before centrifugation) forms a gel-like or white pellet on side and bottom of the tube.

RNA Wash 1.Remove the supernatant and wash the RNA pellet by vortexing with 75 % cold ethanol and subsequent centrifugation at full speed for 5 min at 4 C. Add at least 75% ethanol 1 ml per 1 ml TRI Reagent used fort he initial homogenizaiton. If the RNA pellets accumulates on the side of the tube or has a tendency to float, sediment the pellet at 1200 g.

RNA Solubilization

1. Remove the ethanol wash and briefly air-dry the RNA pellet for 3-5 min. It is important to avoid completely drying the RNA pellet as this well greatly decrease its solubility.
2. Dissolve the pellet with 30 ul RNAase free moleculer grade water.