This document provides instructions for isolating total RNA from tissue using TRIZOL reagent. DEPC-treated water is used to avoid RNase contamination. TRIZOL reagent contains phenol and is toxic, requiring protective gloves and clothing. Tissue is homogenized in TRIZOL, then chloroform is added to separate the mixture into aqueous, interphase, and organic layers. The aqueous layer containing RNA is removed, isopropanol is added to precipitate the RNA, and the RNA pellet is washed with ethanol before being dissolved in DEPC-treated water.

1. ISOLATION OF TOTAL RNA USING TRIZOL REAGENT
DEPC (diethyl pyrocarbonate) is added to water to destroy any RNases that may be present. It is
added to distilled water at a ratio of 0.1% v/v (1mL/1000mL) and allowed to sit overnight at room
temperature. The water is then autoclaved to destroy any lingering DEPC (most of it had probably
degraded overnight). The water can then be used to make up stock solutions or dissolve RNA
samples.
--Gloves must be worn during all manipulations. All tips and tubes must be RNAse free---
NOTE: TRIZOL REAGENT IS TOXIC. This reagent contains pbenol, which will cause burns if it
comes in contact with skin. If contact occurs, wash the area with lots of water and detergent.
1. Weight 50 - 100 mg of the desired tissue in the appropriate manner and place it in a 50 ml
Oak Ridge tube kept in liquid nitrogen or on dry ice. The tissue does not need to be
crushed or broken into smaller pieces.
2. Add 1 ml of Trizol reagent to the Oak Ridge tube. The solution will freeze solid once added.
Warm the tube in your hands until the solutions begins to thaw then homogenize with the
polytron or tissuemizer. For every extra 50 -100 mg of tissue used, use another
milliliter of reagent and another aliquot of chloroform and isoamyl alcohol. For
example, if you weigh out 385 mg of tissue, use 4 ml of Trizol and 4 times the given
amounts of chloroform and isoamyl alcohol.
3. The homogenized sample is allowed to sit at room temperature for ~5 minutes (while you
homogenize your other samples). Add 200 µl (adjust based on starting amount of Trizol) of
chloroform to the sample and shake vigorously for 15 seconds. Allow the sample to
incubate at room temperature for an additional 2 -3 minutes.
4. Centrifuge at 12 000 xg (about 10 000 rpm) in the Sorvall at 4
o
C for 15 minutes.
Centrifugation separates the sample into three layers: a lower organic layer, which
contains proteins; an interphase layer, which contains DNA; an upper aqueous layer, which
contains RNA.
5. Remove the upper aqueous layer using a pipetteman (without disturbing the interface) and
transfer it into a sterile Eppendorf tube. If you think you carried over any of the interphase or
organic layer, spin the tube in the Eppendorf centrifuge for one minute. This will pellet any
sediment and reseparate any layers. Transfer the aqueous phase to a new tube. Add an
equal volume of isopropanol to the tube, invert twice then let incubate at room temperature
for 10 minutes. This is the RNA precipitation step.
6. Pellet the RNA by centrifuging the samples at 12 000 xg for 10 minutes in an Eppendorf
centrifuge at 4
o
C. The pellet should be white. Pour off the supernatant. Add 1 mL of 70%
ethanol to the pellet to remove any lingering chemicals, invert the tube twice then spin at
7500 xg in an Eppendorf centrifuge for 5 minutes. The pelleted RNA can be dissolved in
DEPC-treated water. It is best to store the RNA at -80°C.