Oneill Lab:Trizol

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Michael J. O'Neill Lab University of Connecticut Department of Molecular and Cell Biology

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Contents

Trizol RNA Extraction

Introduction

TRIzol Reagent is a product of Invitrogen for use in total RNA extraction. The manufacturer suggests that DNA and protein can subsequently be extracted. However, we do not use the reagent for this purpose. This protocol is limited to just the RNA extraction.

TRIzol is acidified phenol with guanidine isothiocyanate. Low pH phenol keeps DNA in the organic phase while RNA is extracted from the aqueous. Guanidine isothiocyanate is a chaotropic agent used to rapidly eliminate enzyme activity.

Sample Preparation

Any tissues to be extracted should be snap frozen in liquid nitrogen immediately after dissection. They can be stored indefinitely in a -80°C freezer until needed for RNA. Avoid allowing the sample to thaw prior to extraction. If dealing with a large piece of tissue, it can be stored in pieces to prevent multiple freeze-thaw cycles. TRIzol reagent will also work with cell pellets, which must also be stored at -80°C.

TRIzol Extraction

Tissue Homogenization

  1. Place tissue in a sterile polypropylene culture tube
  2. Add 1 ml TRIzol reagent / 100 mg tissue
  3. Homogenize on Polytron

Phase Separation

  1. Incubate homogenized samples at room temp for 5 minutes
  2. Add 0.2ml chloroform / 1 ml TRIzol used
  3. Securely cap tubes and shake vigorously for 15 seconds
  4. Incubate at room temp for 3 minutes
  5. Centrifuge at 4°C for 15 minutes at 12,000 x g.
  6. Remove aqueous phase to a microfuge tube and precipitate.

RNA Precipitation

  1. Add 0.5ml RNase free isopropanol / 1 ml TRIzol initially used.
  2. Incubate at -20°C for 20 minutes
  3. Centrifuge at 4°C for 10 minutes at 12,000 x g.
  4. Remove supernatant
    • Pellet may be clear or gel-like. Be careful not to loosen or disturb.
  5. Wash with 1ml 70% ethanol / 1 ml TRIzol.
  6. Vortex briefly
  7. Centrifuge at 4°C for 5 minutes at 7,500 x g.
  8. Remove ethanol and air dry pellet
    • Do not let pellet dry completely
  9. Resuspend in DEPC treated water
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