RNA extraction from colonial tunicates

Clean the slide from which you will take the colony of your interest. See Cleaning colonial ascidians.

Isolate a cleaned colony composed of approx. 20 zooids.

Transfer to a tube and spin at maximum speed for 0h 2m 0s .

Remove the excess water and shock-freeze the tube in liquid nitrogen.

Add 400µL of extraction buffer to the frozen sample and macerate with a plastic pestle.

Add 100µL more of extraction buffer and 500µL of 1:1 phenol:chloroform.

Mix the tube by inversion a couple of times until it gets cloudy.

Centrifuge the homogenate at 1400 g for 0h 5m 0s at 4°C .

Carefully collect 400µL of the upper phase into a new tube.

Note: if desired this sample could be used for DNA extraction - carefully transfer 200µLof the interphase into a new tube (See DNA extraction from colonial tunicates).

Add 500µL of 6Molarity (M) LiCl to the supernatant.

Incubate the mixture at -80°C for 1h 0m 0s .

Centrifuge at 1400 g for 0h 10m 0s at 4°C .

Discard the supernatant and resuspend the pellet in 1mL of 3Molarity (M) LiCl.

Shake slowly for 0h 15m 0s at Room temperature on a linear shaker.

Centrifuge at 1400 g for 0h 10m 0s at 4°C .

Discard the supernatant and resuspend the pellet in 1mL of SC-EtOH solution.

Incubate at -80°C for 0h 15m 0s .

Centrifuge at 1400 g for 0h 15m 0s at 4°C .

Discard the supernatant and wash the pellet with 1mL of 70% volume Ethanol.

Centrifuge at 1400 g for 0h 5m 0s at 4°C .

Discard the supernatant and place the tubes up-side-down on a paper towel for 0h 5m 0s to 0h 10m 0s .

Resuspend the pellet in RNase-free water (20µL to 100µL depends on the amount of pellet).

Quantify the RNA concentration and quality using the NanoDrop, the capillary electrophoresis and/or the Bioanalyzer.

Store at -80°C .