RNA extraction using Trizol

From ChoanoWiki

Trizol RNA extraction protocol

This method may be used for difficult samples where RNA yield from kits (e.g. RNeasy) is low

1.

• Pellet cells by centrifuging at 2700g for 40 minutes at 4^oC
• Carefully remove the supernatant
• Add 500μl ice-cold PBS and vortex

2.

• Centrifuge at 300g for 5 minutes
• Add 1ml Trizol reagent

3.

• Repeatedly syringe the cells through a 0.8mm needle (at least 10 passages)
• Incubate at room temperature for 5 minutes

4.

• Add 0.2ml chloroform and vortex
• Allow to settle for 2-3 minutes
• Centrifuge at 12000g for 15min at 4^oC

5.

• Transfer upper aqueous layer to a sterile eppendorf tube
• Repeat step 4 until the boundary layer is clean and free of white precipitate

6.

• Add 0.5ml isopropanol
• Incubate at room temperature for 10 minutes
• Centrifuge at 12000g for 10 minutes at 4^oC

7.

• Carefully remove the supernatant.
• Add 1ml 70% Ethanol and vortex
• Centrifuge at 7500g for 5 minutes at 4^oC

8.

• Carefully remove the supernatant ethanol
• Allow the pellet to air dry
• Resuspend in 30μl RNase-Free sterile water
• Run a sample of the RNA on a 1% agarose gel to verify that intact RNA has been extracted

Footnote: Protocol posted by Alan Marron July 2009