Jan 28, 2022

Public workspaceRNA extraction from colonial tunicates

DOI
dx.doi.org/10.17504/protocols.io.b33nqqme
  • 1University of Fribourg
  • Blanchoud lab, UNIFR
Marta Wawrzyniak
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DOI: dx.doi.org/10.17504/protocols.io.b33nqqme
Protocol Citation: Marta Wawrzyniak, Simon Blanchoud 2022. RNA extraction from colonial tunicates. protocols.io https://dx.doi.org/10.17504/protocols.io.b33nqqme
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: January 20, 2022
Last Modified: January 28, 2022
Protocol Integer ID: 57166
Keywords: ascidians, colonial tunicates, RNA extraction
Abstract
This protocol has been successfully used with Botrylloides diegensis and has been adapted from the following publication:

An efficient low-cost laboratory workflow for the study of blood cells and RNA extractions in marine invertebrates
Guidelines
Change gloves frequently, particularly as the protocol progresses from crude extracts to more purified materials. Use sterile tubes. Perform all steps on ice and use RNAse-free water unless otherwise stated.
Materials
Liquid nitrogen
Sterile tubes and plastic pestles
Extraction buffer : 0.2M Tris-HCl pH 7.5 , 0.1M LiCl, 5mM EDTA, 1/10 of the total volume of SDS 10%
Phenol pH 4 (4 C)
Chloroform
LiCl (for 50mL: 12.6g 6M LiCl ; 6.3g 3M LiCl)
SC-EtOH: Sodium acetate + 100% Ethanol (1/3 : 2/3)
70% and 100% Ethanol
RNase-free water
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 Duration00:02:00 .

2m
Remove the excess water and shock-freeze the tube in liquid nitrogen.
Add Amount400 µL of extraction buffer to the frozen sample and macerate with a plastic pestle.

Add Amount100 µL more of extraction buffer and Amount500 µL 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 Duration00:05:00 at Temperature4 °C .

5m
Carefully collect Amount400 µL of the upper phase into a new tube.

Note: if desired this sample could be used for DNA extraction - carefully transfer Amount200 µL of the interphase into a new tube (See DNA extraction from colonial tunicates).

Add Amount500 µL of Concentration6 Molarity (M) LiCl to the supernatant.

Incubate the mixture at Temperature-80 °C for Duration01:00:00 .

1h
Centrifuge at 1400 g for Duration00:10:00 at Temperature4 °C .
10m
Discard the supernatant and resuspend the pellet in Amount1 mL of Concentration3 Molarity (M) LiCl.

Shake slowly for Duration00:15:00 at TemperatureRoom temperature on a linear shaker.

15m
Centrifuge at 1400 g for Duration00:10:00 at Temperature4 °C .
10m
Discard the supernatant and resuspend the pellet in Amount1 mL of SC-EtOH solution.

Incubate at Temperature-80 °C for Duration00:15:00 .

15m
Centrifuge at 1400 g for Duration00:15:00 at Temperature4 °C .
15m
Discard the supernatant and wash the pellet with Amount1 mL of Concentration70 % volume Ethanol.

Centrifuge at 1400 g for Duration00:05:00 at Temperature4 °C .
5m
Discard the supernatant and place the tubes up-side-down on a paper towel for Duration00:05:00 to Duration00:10:00 .

15m
Resuspend the pellet in RNase-free water (Amount20 µL to Amount100 µL depends on the amount of pellet).

Quantify the RNA concentration and quality using the NanoDrop, the capillary electrophoresis and/or the Bioanalyzer.
Store at Temperature-80 °C .