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RNA-Bee
ISOLATION OF RNA
U.S. PATENT For in vitro research use TEL-TEST BULLETIN NO. 3
PRODUCT: RNA-Bee - RNA ISOLATION REAGENT
Catalog No: CS-104B - 100 ml / CS-105B - 200 ml / CS-501B - 500
ml
Storage: Store at 2 - 8 C.
PRODUCT DESCRIPTION
RNA-Bee is a complete and ready-to-use reagent for isolation of
total RNA from samples of human, animal, plant, bacterial and viral
origin. RNA-Bee is the improved version of the single-step method
of RNA isolation (1). The improved RNA-Bee provides a fast and highly
reliable method for isolating pure and undegraded RNA from a large
variety of biological samples.
RNA-Bee and the single-step method are subjects of the US patent
4,843,155.
RNA-Bee is a monophase solution containing phenol and quanidine
thiocyanate. A biological sample is homogenized or lysed in RNA-Bee
and the homogenate/lysate is separated into aqueous and organic
phase by the addition of chloroform. The subsequent centrifugation
efficiently removes DNA and proteins from the aqueous phase containing
RNA. The undegraded, pure RNA is obtained from the aqueous phase
by the isopropanol precipitation, washing with ethanol and solubilization
in an appropriate solution, The entire isolation procedure can be
completed in 1 hour. The isolated RNA is appropriate for Northern
blotting, poly A + selection, RT-PCR, and other molecular biology
techniques.
STABILITY: RNA-Bee is stable at 2 - 8 C for at least one year from
date of purchase.
SPECIAL HANDLING PRECAUTIONS
RNA-Bee contains a poison (phenol) and an irritant (guanidine thiocyanate).
CAUSES BURNS. Can be fatal. When working with RNA-Bee, use gloves
and eye protection (face shield, safety goggles). Do not get on
skin or clothing. Avoid breathing fumes. Read the warning note on
the container and MSDS. In case of contact: Immediately flush eyes
or skin with a large amount of water for at least 15 minutes and
seek medical attention.
I. PROTOCOL FOR RNA ISOLATION
Reagents required but not supplied: chloroform isopropanol, and
ethanol.
We recommend the use of disposable polypropylene tubes. The tubes
should be tested to ensure integrity during centrifugation at 12,000g
with RNA-Bee and chloroform.
The protocol describes isolation of RNA with 1 ml of RNA-Bee using
the following steps:
1. HOMOGENIZATION 1 ml RNA-Bee + 50 mg tissue or 5 x 10^6 cells
2. PHASE SEPARATION homogenate + 0.2 ml chloroform
3. RNA PRECIPITATION aqueous phase + 0.5 ml isopropanol
4. RNA WASH 1 ml 75% ethanol
5. RNA SOLUBILIZATION Water, 0.5 % SDS or buffer
All steps can be carried out at room temperature unless otherwise
stated.
1. HOMOGENIZATION
A. TISSUES. Homogenize tissue samples in RNA-Bee (1 ml / 50 mg
tissue) using a glass-glass, glass-teflon, or Polytron homogenizer.
The sample volume should not exceed 10% of the RNA-Bee volume.
B. CELLS. Cells grown in monolayer should be lysed directly in the
culture dish by the addition of RNA-Bee. Use at least 1 ml of the
reagent for a 3.5 cm petri dish. Pass the lysate through a pipette
several times to ensure lysis. Cells grown in suspension should
be sedimented first and then lysed by the addition of RNA-Bee. Add
at least 0.2 ml of RNA-Bee per 10^6 cells and lyse by repeated pipetting.
2. PHASE SEPARATION
Add 0.2 ml chloroform per 1 ml of RNA-Bee, cap the tube and shake
vigorously for 15 - 30 seconds. Store the sample on ice (or at least
4 C) for 5 minutes. Centrifuge the homogenate at 12,000g for 15
minutes at 4 C. Following centrifugation, the sample forms the lower
blue phenol-chloroform phase, interphase, and the upper colorless
aqueous phase. RNA remains exclusively in the aqueous phase whereas
DNA and proteins are in the interphase and organic phase. The volume
of the aqueous phase is about 50% of the initial volume of RNA-Bee
plus sample volume.
Chloroform should not contain isoamyl alcohol or any other additives.
3. RNA PRECIPITATION
Transfer the aqueous phase to a clean tube, add 0.5 ml of isopropanol,
and store the sample for 5-10 minutes at room temperature. Centrifuge
at 12,000g for 5 minutes at 4 - 25 C. RNA precipitate (often not
visible before centrifugation) forms a white-yellow pellet at the
bottom of the tube.
4. RNA WASH
Remove the supernatant and washs the RNA pellet once with 75% ethanol,
shaking or votexing to dislodge the pellet from the side of the
tube. Centrifuge for 5 minutes at 7,500g at 4 - 25 C. Use at least
1 ml of ethanol solution per 1 ml of RNA-Bee used for the initial
homogenization.
An additional wash with 75% ethanol improves 260/280 ratio and might
be necessary to use the isolated RNA in enzymatic assays.
5. RNA SOLUBILIZATION
At the end of the procedure, briefly air-dry the RNA pellet (5 -
10 minutes). It is important not to let the RNA pellet dry completely,
as this greatly decreases its solubility. Do not dry RNA by centrifugation
under vacuum. Dissolve the RNA in water, 0.5% SDS or buffer by passing
the solution through a pipette ip and/or incubating for 10 - 15
minutes at 55 - 60 C. Tubes, water or solutions used for RNA solubilization
should be made RNase-free by diethyl pyrocarbonate (DEPC) treatment.
The final preparation of RNA has a 260/280 ratio 1.6 - 1.9.
II. COMMENTS
1. Isolation of RNA from a small amount of tissue (1-10 mg) can
be accomplished by homogenizing the sample in 0.8 ml of RNA-Bee.
Transfer the homogenate to an Eppendorf tube, add 160 l of chloroform,
and store the sample for 5 minutes at 4 C. Centrifuge for 15 minutes
at 4 C, collect the aqueous phase and precipitate the RNA with 0.4
ml of isopropanol for 30 minutes or overnight at 4 C. Centrifuge
RNA precipitate at 10,000g for 10 minutes at 4 - 25 C. Wash the
pellet once with 75% ethanol.
2. Following isopropanol addition, store the sample overnight at
4 C in case the procedure has to be interrupted at this step.
3. Hands and dust are a significant source of RNase contamination.
Use gloves and keep tubes clean.
4. Some commercial SDS preparations have an acidic pH. Adjust pH
to 6.5 - 7.5 if necessary.
III. REFERENCES
1. P. Chomczynski and N. Sacchi, Anal. Biochem. 162, 156-159 (1987).
RNA-Bee is a trademark of Tel-Test, Inc.
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