RNA Isolation with TRIzol (Invitrogen) and Qiagen RNAeasy

This protocol applies to: Neuroblastoma (NBL; prior to 2013 only)

The protocol herein describes the procedures used by Nationwide Children’s Hospital to
process disease tissues for RNA and/or DNA subsequently used for characterization in
the NCI’s TARGET initiative. All nucleic acid samples used in TARGET projects were
quality tested for consistency using picogreen quantification and SSTR genotyping
methods, regardless of where the nucleic acid was originally extracted.

I. PRINCIPLE
The Invitrogen Life Technologies TRIzol Reagent (Total RNA Isolation Reagent)
is a ready-to-use reagent for the isolation of total RNA from cells and tissues for
use in PCR analysis. TRIzol reagent is a mono-phasic solution of phenol and
guanidine isothiocyanate. During tissue homogenization or lysis, the TRIzol
reagent maintains RNA integrity, while disrupting cells and dissolving cell
components. Addition of chloroform followed by centrifugation separates the
solution into an aqueous phase and an organic phase. RNA remains in the
aqueous phase and is recovered by precipitation with isopropanol. The RNA is
redissolved in Nuclease-free water. Patient RNA is stored for research requests.

II. SPECIMEN
A. Type
Solid tumor tissue

B. Handling Conditions
Standard Precautions must be followed when handling all solid tissue
samples. Samples must be stored in a -80C freezer.

C. Sample Preparation
The solid tumor tissue may be fresh or frozen. Fresh tumor is suboptimal
due to likely RNA degradation. Optimum sample size is a small cube of
tissue weighing between 50-70 mg. Testing will be attempted on all
samples, irrespective of percentage of viable tumor. However, if there is
less than 30% viable tumor and the testing results are negative, no result
will be reported.

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 D. Indications for Study
RNA isolation is necessary for any assays which analyze the presence or
quantity of mRNA transcripts, e.g. RT-PCR.

III. REQUIRED EQUIPMENT, SUPPLIES AND REAGENTS

A. Equipment
Capsule Centrifuge
Magnetic Stir Plate
Microcentrifuge (Eppendorf 5415R)
Omni H Homogenizer, 115V, 60Hz or TissueLyser (120V, 50/60 Hz)
Pipetters - Adjustable, 1-10 L, 5-20 L, 20-200 L, 100-1000 L
Nanodrop
Vortex Mixer
Water Bath

B. Supplies
Aerosol Barrier Pipet Tips
Disposable Homogenizers
Dry ice
Forceps
PPE (Gloves, Lab Coat)
50 mL Serological Pipette
Ice
Labels (TEC, 2.625”x1” and 2”x0.625”)
0.5 mL Micrewtube Tubes, screw cap (Milian, T338-2S)
mL Micrewtube Tubes, screw cap (Milian, T338-5S)
Yellow cap inserts (Milian, TIL-21)
Petri dishes (35x10 mm)
50 mL Polypropylene Centrifuge Tube
Scalpel blades
2 mL Screw-cap Polypropylene Microcentrifuge Tubes
2 mL Safe-Lock Tubes (Eppendorf, Cat# 2236335-2)
Stainless Steel Beads, 5 mm (200) (Qiagen, Cat# 69989)
Tube Racks

C. Reagents
Chloroform (Sigma, Catalog # C2432)

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 Nuclease Free Water (Fisher, Catalog # BP2484-50)
75% Ethanol (-20C) (Sigma, Catalog # E7023)
Isopropanol (Sigma, Catalog # I0398)
TRIzol reagent (Invitrogen Life Technologies, Catalog # 15596-026)
DNA-free Kit, includes DNAse I buffer, DNase, Inactivation reagent
(Ambion, Catalog #1906)

IV. WARNINGS / PRECAUTIONS

A. Use Standard Precautions when handling all body fluids, tissues and cell
cultures. Refer to the Specimen Collection and Handling, GEN-1, for guidelines
specific for the Molecular Genetics Laboratory and samples.
B. TRIzol reagent is toxic when in contact with skin and if swallowed. It will cause
burns. Be sure to wear a lab coat, gloves and safety glasses when working with
TRIzol reagent. If in contact with skin, wash immediately with plenty of soap and
water. Work in a chemical fume hood.
C. To prevent RNase contamination, always wear gloves and change them
frequently. Also, use sterile, disposable plasticware and pipettes dedicated
strictly to RNA work to prevent cross-contamination with RNases from shared
equipment.
D. RNA is extremely susceptible to degradation by ribonucleases that are ubiquitous
in the environment. To ensure preservation of target RNA or RNA probes,
special precautions are needed. Records are maintained to show that RNase-
free conditions (i.e. wiping the lab areas with RNaseZAP) are met, with corrective
action if conditions are not met.

V. REAGENT PREPARATION (INCLUDING STORAGE

CONDITIONS)
A. TRIzol reagent is used as supplied by Invitrogen Life Technologies. It is stored in
a refrigerator in a dark container.
B. Chloroform and isopropanol are stored at room temperature in the flammable
liquids chemical cabinet.
C. DNA-free kit from Ambion is stored in a -20oC freezer.

D. 75% ethanol
1. To a 50 mL polypropylene centrifuge tube, add 37.5 mL of 100% ethanol.
Next add distilled water to reach a final volume of 50 mL. Invert to mix.

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 2. Label tube appropriately (reagent name, date made, expiration date,
storage conditions, NFPA rating and initials) and be sure to mark for RNA
USE ONLY. Store in the flammable cabinet.
3. Write preparation information in to the QC Book, under the Prepared
Reagents tab.

VI. QUALITY CONTROL

A. Specimen identification is assured through each phase of handling by
assignment of a specific color to each sample. That color (in addition to the lab
number and patient initials) is placed on each tube used throughout the
procedure.
B. At each step in the isolation, the supernatant or pellet that does not contain the
RNA is retained until after isolation and quantification is completed. All tubes are
labeled with the COG number, BPC number and patient initials.

VII. PROCEDURE – STEPWISE (Follow worksheet for RNA

Isolation)
A. Homogenization with TissueLyser
(NOTE: Work with one specimen at a time until all specimens are in TRIzol)
1. Place a 5 mm stainless steel bead in a Safe-Lock tube for each specimen.
2. In a chemical fume hood, add 0.5 mL of TRIzol reagent to an appropriately
labeled 2 mL Safe-Lock Eppendorf tube and place on wet ice.
3. Frozen tissue should remain buried in dry ice until added to Trizol. The
optimum sample size is between 50-70 mg; if over 70 mg, split the sample
into two pieces and put into 2 SafeLock tubes (combine these after
homogenization).
4. Place the entire tumor into the Safe-Lock tube containing TRIzol.
5. Once all of the samples are in their labeled tubes, place the tubes into the
TissueLyser adapter sets.
6. Fix the adapter sets into the clamps (arms) of the TissueLyser.
Homogenize the samples for 3 minutes at 25 Hz. If the sample is not fully
homogenized, repeat with decreased time and decreased frequency (1
minute, 20 Hz) for no more than 2 additional minutes. For optimal
operation, the TissueLyser must always be balanced.
7. Working in a chemical fume hood, transfer the homogenized sample by
pipet to a new 2 mL screw cap tube containing 0.5 mL of TRIzol. Invert
tube to mix. Place the tube on wet ice temporarily until ready to proceed to

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 Phase Separation, or on dry ice and then move into a -80° freezer for
storage of homogenized sample for future extraction.

B. Phase Separation
1. Incubate the homogenized sample for 5 minutes at room temperature. (If
tubes are frozen, thaw in a bucket of wet ice before proceeding to room
temperature incubation.)
2. In a chemical fume hood, add 0.2 mL of chloroform per 1 mL of TRIzol
reagent.
3. Cap sample tubes securely and shake vigorously by hand for 15 seconds.
Incubate the samples at room temperature for 3 minutes.
4. Following the incubation, centrifuge the samples at 11,600 x g for 15
minutes at 4C.
5. Following centrifugation, the mixture separates into a lower red phenol-
chloroform phase, an interphase and a colorless, upper aqueous phase.
RNA remains exclusively in the aqueous phase (which is about 60% of the
volume of TRIzol reagent used for initial homogenization). However, the
organic phase can be saved for subsequent DNA and protein extraction.

C. RNA Precipitation
1. Add 0.5 mL of isopropanol per 1 mL of TRIzol reagent originally used to a
new tube.
2. Transfer the aqueous phase to the labeled isopropanol tube. Precipitate
the RNA from the aqueous phase by pipetting up and down gently. (Do
not vortex.)
3. Incubate the samples for 10 minutes at room temperature, then centrifuge
at 11,600 x g for 10 minutes at 4C.
4. The RNA precipitate forms a translucent gel-like pellet on the side and
bottom of the tube.

D. RNA Wash
1. Decant the supernatant to a clean, labeled 1.5 mL microcentrifuge tube
and set aside.
2. Wash the RNA pellet once with 75% ice-cold ethanol. Use 1 mL of 75%
cold ethanol per 1 mL of TRIzol reagent used.
3. Mix by inversion. Do not vortex. Centrifuge at 10,000 x g for 5 minutes at
4C.

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 E. Resuspending the RNA
1. Remove supernatant with a pipet. Quickly spin in a capsule centrifuge to
collect any remaining 75% ethanol to the bottom. Remove as much of the
remaining ethanol with a pipet and air dry the RNA pellet by leaving the
tubes open on the counter for approximately 15-30 minutes. When the
pellet is dry, there must be no visible ethanol in the tube. Do not over dry
the pellet as it may be difficult to redissolve.
2. Resuspend the RNA pellet in 50 µL of nuclease-free water by pipetting up
and down gently and incubating in a 60C water bath for 10 minutes (can
incubate longer if necessary, up to one hour). Flick (do not vortex) gently
to mix and quickly spin to collect the solution.

F. DNase Treatment of RNA

1. Add 0.1 volume (5.6 µL) of 10X DNase I buffer and 1 µL of DNase I (2
units) to the RNA. Mix gently by flicking tube (DO NOT VORTEX) and
incubate in a 37oC water bath for 20 minutes.
2. Add 0.1 volume of DNAse Inactivation Reagent (6.17 µL) to the RNA.
(Make sure to vortex the DNAse Inactivation Reagent before addition to
the RNA.) Flick (do not vortex) the RNA tube to disperse the reagent.
Incubate for 2 minutes at room temperature. Flick the tube once more
during the incubation to redisperse the DNase Inactivation Reagent.

3. Centrifuge at 10,000 x g for 1 minute at 4C. Transfer the supernatant

RNA solution to a 1.5 mL micrewtube labeled with the BPC number,
patient initials, and parent ID or barcode.

G. Determination of RNA Concentration

Quantitate RNA by measuring absorption at 260 nm – refer to the Quantitation of
Nucleic Acids procedure, GEN-2.

VIII. INTERPRETATION / ANALYSIS / DOCUMENTATION

A. For each sample, enter the following information into the extraction Log (internal
document): BPC number, COG number, patient initials, Parent ID or barcode,
volume, A260 reading, A280 reading, A260/280 reading, concentration in µg/mL,
QC volume, patient ID, live tumor %, tumor %, initials, date of isolation and
comments

B. Yields from solid tissue are generally greater than 25 g RNA per 50 mg of solid
tissue. The yield will vary depending on the numbers of white blood cells
present.

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 C. Aliquot 2µL RNA into a labeled 0.5 mL Eppendorf tube for QC purposes. QC’ing
is done using REF-30, QC for Nucleic Acids. The RIN value is logged into the
RNA spreadsheet.

D. RNA is currently stored in a -80°C freezer indefinitely.

IX. REFERENCES
1. TRIzol reagent literature, Invitrogen Life Technologies.
Some samples have been cleaned-up using the QIAGEN RNAeasy Clean-up Kit.