*Faster 1 hour protocol
*Blue dye added to reagent to facilitate organic/aqueous
*Applications are the same as those of RNAzol
*2 ml isolates RNA from 100 mg tissue or 1 x 107 cells or 10
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 -
Storage: Store at 2 - 8 C.
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
RNA-Bee and the single-step method are subjects of the US
RNA-Bee is a monophase solution containing phenol and
guanidine 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,
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.
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
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
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.
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.
1. P. Chomczynski and N. Sacchi, Anal. Biochem. 162, 156-159
RNA-Bee is a trademark of Tel-Test, Inc.