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PRODUCT DESCRIPTION
Recent progress in RNA isolation
technology has made it possible to replace lengthy and laborious
methods of total RNA isolation (1) by a single-step method (2,3).
The RNA STAT-50TM LS is a new and
substantially improved version of the single-step method. It is a
complete and ready to use reagent for isolation of total RNA from
liquid samples of human, animal, plant, yeast and bacterial origin.
Extensive laboratory tests have shown that RNA STAT-50 TM LS is highly reliable and produces very
consistent results.
The composition of RNA STAT-50TM LS includes phenol and guanidinium
thiocyanate in a mono-phase solution. A biological specimen is lysed
(or homogenized) in RNA STAT-50TM LS
and mixed with chloroform. Following centrifugation, the lysate
separates into two phases: aqueous phase and organic phase. The
total RNA remains exclusively in the aqueous phase while DNA and
proteins are extracted into an organic phase and interphase. The
total RNA is precipitated from the aqueous phase by addition of
isopropanol, washed with ethanol and solubilized in water. RNA
STAT-50 TM LS is the most effective
method of RNA isolation from biological fluids such as blood, serum,
amniotic fluid, CSF etc. The entire procedure can be completed in
1 hour and the recovery of undegraded mRNAs I s30-150% greater than
with any other method of RNA isolation.
APPLICATION
The total
RNA isolated by RNA STAT-50TM LS is undegraded and
free of DNA contamination. It can be used for Northern analysis, dot
blot hybridization, poly A+ selection, in vitro
translation. RNase protection assay, molecular cloning, and for
polymerase chain reaction (PCR) without treatment with DNase or
additional purification. The simplicity of the isolation using RNA
STAT-50 TM LS makes it possible to
process simultaneously a large number of samples, and the excellent
recovery of RNA permits the use of this product for isolation of RNA
from very small biological specimens.
PROTOCOL
Reagent supplied: RNA STAT-50TM LS.Reagents required,
but not supplied: chloroform, isopropanol and ethanol.
The methods include the
following steps:
1. Homegenization 0.75 ml RNA STAT-50TM + 0.25 ml sample.
2. RNA Extraction homogenate +0.2 ml chloroform.
3. RNA Precipitation aqueous phase +0.5 ml isopropanol.
4. RNA Wash 1 ml 75% ethanol.
Unless stated otherwise the procedure is carried out at room
temperature. The use of sterile, disposable polypropylene tubes is
recommended throughout the procedure. Before using test if the tubes
can withstand centrifugation at 10,000 x g with the RNA
STAT-50TM
LS.
1. HOMOGENIZATION
A. BIOLOGICAL FLUIDS.
Mix 0.75
ml of RNA STAT-50TM LS with 0.25 ml of sample
and lyse cells (or
cellular debris) suspended in the sample by passing the mixture
several times
through a pipette. Use at least 0.75 ml of RNA STAT-50TM LS per
5-10 x 106
cells.
B. TISSUE SUSPENSIONS.
Homogenize
0.25 ml sample with 0.75 ml of RNA STAT-50TM LS in a glass-Teflon
or Polytron homogenizer. If the sample vol. is < 0.25 ml, adjust
the volume
with water. The volume ration of RNA STAT-50TM
LS
to sample should always be 3 to 1.
2. RNA EXTRACTION
Following homogenization, store the
homogenate for 5 minutes at room temperature
to permit the complete dissociation of nucleoprotein complexes.
Next, add 0.2 ml of chloroform per 0.75
ml of RNA STAT-50TM
LS,
cover the samples tightly, shake vigorously for 15 seconds and let
them stay at room temperature for 2-3 minutes,
centrifuge the homogenate at 12,000 g (max.)
for 15 minutes at 4oC. Following centrifugation, the homogenate
separates into two phases: a lower red, phenol-chloroform
phase and the colorless upper aqueous phase
whereas DNA and proteins are in the inter phase and
organic phase. The volume of the aqueous phase is about 60% of the
volume of homogenate.
3. RNA PRECIPITATION
Transfer the aqueous phase to a fresh
tube and mix with isopropanol. Add 0.5 ml of
isopropanol per 0.75 ml of RNA STAT-50 TM
LS
for homogenization. Store samples at room temperature for 5-10 min
and centrifuge at 12,000 g (max.) for 10 minutes
at 4oC. RNA precipitate (often invisible
before centrifugation) forms a white pellet at the bottom of the tube.
4. RNA WASH
Remove supernatant and wash the RNA
pellet once with 75% ethanol by vortexing
and subsequent centrifugation at 7,500 g (max.) for 5 minutes at
4oC. Add at least 1 ml of 75%
ethanol per 0.75 ml of RNA STAT-50TM LS used for the
initial homogenization.
At the end of the procedure, dry briefly
the RNA pellet by air-drying or under vacuum
(5-10 min). It is important not to let the RNA pellet dry completely
as this will greatly decrease its solubility.
Do not use the Speed-Vac for drying. Dissolve
the RNA pellet in FORMazolTM (Cat. # T-4025), water
or in 0.5% SDS solution. Vortex or pass
the pellet a few times through a pipette tip. An incubation
for 10-15 minutes at 55-60oC is required to completely
dissolve RNA samples. The SDS solution used
for RNA solubilization should be made free
of RNase by diethyl pyrocarbonate (DEPC) treatment.
NOTE: Should you use our RNase free purified water (Cat. # T1-3200)
this treatment
step is unnecessary. The final preparation of total RNA
is free
of DNA and proteins and has a 260/280 ration > 1.8.
NOTES AND COMMENTS
1. Isolation of RNA from small samples
(<106 of cells). Lyse (or homogenize) samples
in 0.75 ml of RNA STAT-50TM
LS in the Eppendorf tube
and follow the isolation
protocol with the exception of the RNA precipitation which should
be carried out for 30 minutes at 4oC. If the expected
yield of RNA is < 1
ug, an addition of 5 ug of carrier tRNA or glycogen (molecular biology
grade, Boehringer
Mannheim) to the aqueous phase is necessary for a 100% recovery
of the RNA precipitate.
2. Following homogenization (before addition
of chloroform) samples can be stored
at -70oC for at least two weeks.
3. Hands and dust may be the major source
of the RNase contamination. Use gloves
and keep tubes closed throughout the procedure.4. Citation of the
method: total RNA
was isolated by the single-step method (2) using RNA STAT-50TM LS.
SPECIAL HANDLING PRECAUTIONS
The RNA
STAT-50TM LS contains poison
(phenol) and irritant (guanidinium thiocyanate). CAN BE FATAL. When
working with RNA STAT-50TM LS use gloves and
eye protection (shield, safety goggles). Do not get on skin or
clothing. Avoid breathing vapor. Read also the warning note on the
bottle.
In case of contact: Immediately flush eyes or skin with a large
amount of water for at least 15 minutes and seek immediate medical
attention.
REFERENCES
1. Sambrook J.,Fritsch E.F. and Maniatis
T. (1989) Molecular Cloning. Cold Spring
Harbor Laboratory, Cold Spring, Harbor, N.Y.
2. Chomczynski P. and Sacchi N. (1987) Single-step
method of RNA isolation by
acid guanidinium thiocyanate-phenol-chloroform extraction. Anal.
Biochem. 162, 156-159.
3. Chomzynski P. (1989) The RNAzolTM B method.
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