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
