Optimized Protocol for RNA Extraction from Insect Samples Using TRIzol Reagent

Cover the crucible with liquid nitrogen to keep the sample cold

Place the sample (about 50mg) and macerate, keeping it cold. Remove the wings and macerate insect samples as they have a strong exoskeleton and the presence of chitin

Mix the sample with Trizol in the crucible and transfer to a 1.5 mL microtube

Add 200 µL of ice-cold chloroform for phase separation. Shake the tube vigorously by hand for a full 15 seconds. Let sit at room temperature for 2-3 minutes

NOTE : You should see the two mixtures separating almost immediately: pink on the bottom and clear on top

Add 1mL of Trizol and wait for it to thaw

Centrifuge at 12000 rpm for 15 min at 4° C

NOTE : Any denatured proteins might appear as a white interface. Avoiding this interface, carefully move the clear top liquid to a new clean, labeled tube

Remove 500 µL of supernatant and add 400 µL of ice-cold isopropanol to precipitate the RNA (always 100 µL less)

Leave 10 minutes at room temperature

NOTE : Leaving to precipitate in the -20°C is not recommended because it might cause contaminants to co-precipitate

Centrifuge at 12000 rpm for 10 min 4° C

NOTE : You may see the pellet as a clear, gel-like ball at the base of the tube, or a small white smear

Remove the isopropanol and wash with 1 mL of 75% ethanol

Centrifuge at 9500 rpm for 5 min at 4° C

Discard the alcohol and wait for the pellet to dry for 5 to 10 minutes at room temperature

NOTE : Remove as much of the ethanol as possible

Resuspend in 30 µL of RNAse-free water depending on the size of the pellet formed