RNA Extraction from Drosophila Tissues using TRIzol Reagent
Add 1 mL TRIzol Reagent to 50 - 100 mg of frozen Drosophila tissue in a 1.5 mL microcentrifuge tube , and homogenize immediately with a disposable plastic pestle .
Centifuge the sample at 12,000 x g for 10 minutes at 4˚C .
* Pellet contains ECM , polysaccharides , and high molecular weight DNA ; supernatant contains the RNA .
In high fat samples , a layer of fat collects above the supernatant .
Remove and discard the fatty layer .
Transfer the cleared supernatant to a new tube .
Incubate the sample for 5 minutes at room temperature .
Add 0.2 mL of chloroform , and cap the tube securely .
Shake the tube vigorously by hand for 15 seconds .
Incubate for 2 - 3 minutes at room temperature .
Centrifuge the sample at 12,000 x g for 15 minutes at 4˚C .
* The mixture separates into a lower red phenol - chloroform phase , an interphase , and a colorless upper aqueous phase .
RNA remains in colorless aqueous phase ( ~ 50 % of the total volume ) .
Remove the aqueous phase of the sample by angling the tube at 45˚ and pipetting the solution out .
Place the aqueous phase into a new tube .
* Avoid drawing any of the interphase or organic layer into the pipette .
* The interphase and organic phenol - chloroform phases can be saved for DNA or protein isolation if desired ( saved overnight at 4˚C ) ; however , the protocols for these procedures will not be discussed here .
Please refer to the manufacturer's TRIzol Reagent manual .
Add 0.5 mL of 100 % isopropanol to the aqueous phase .
Incubate sample at room temperature for 10 minutes .
Centrifuge at 12,000 x g for 10 minutes at 4˚C .
* RNA is often visible prior to centrifugation , and forms a gel - like pellet on the side and bottom of tube .
Remove all supernatant from the tube , leaving the RNA pellet .
Wash the pellet with 1 mL 75 % ethanol .
* RNA can be stored in 75 % ethanol at least 1 year at - 20˚C , or at least 1 week at 4˚C .
Briefly vortex the sample . Centrifuge the tube at 7,500 x g for 5 minutes at 4˚C . Discard the wash .
Vacuum or air dry the RNA pellet for 5 - 10 minutes .
* Do not dry the pellet by vacuum centrifuge .
* Do not allow the RNA to dry completely .
Resuspend the RNA pellet in RNase - free water by passing the solution up and down several times through a pipette tip .
Incubate in a water bath or heat block set at 55 - 60˚C for 10 - 15 minutes .
Proceed to downstream applications , such as DNase treatment or cDNA synthesis , or store at - 70˚C
