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1. INTRODUCTION
Recent progress in RNA isolation technology
has made it possible to replace lengthy
and laborious methods of total RNA isolation1 by a single-step
method2.3 RNA STAT-60TM
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 tissues and cells of human,
animal, plant, yeast, bacterial, and viral origin.
Extensive laboratory tests have shown that the RNA STAT-60 TM
is highly reliable and produces very consistent
results.
The composition of RNA STAT-60TM
(patent pending) includes phenol and guanidinium
thiocyanate in a mono phase solution. A biological sample is homogenized
in the RNA STAT-60TM using a glass-Teflon or Polytron homogenizer.
Upon addition of chloroform, the homogenate 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 waster.
The entire procedure for RNA isolation using
the RNA STAT-60TM can be completed
in 1 hour. This is the most effective method of RNA isolation. The
recovery of undegraded mRNAs using
the RNA STAT-60TM is 30-150% greater
than with any other method of RNA isolation. RNA STAT-60TM offers:
* Total RNA/mRNA in under 60 minutes.
* Northern blot/PCR* - ready
mRNA in under 60 minutes.
* No further purification required for
use in subsequent procedures including
Northern blotting and PCR* .
* Extracts 30-150% more total RNA/mRNA
than any other method.
* Cost effective method requiring less
reagent/sample.
2. APPLICATION
The
total RNA isolated by the RNA STAT-60TM is undegraded and free
of protein and 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 additional treatment
with DNase. The simplicity of the isolation using the RNA
STAT-60TM makes it possible to process
simultaneously a large number of samples,
and the excellent recovery of RNA from very small biological samples
(biopsies, etc.).
3. REAGENTS SUPPLIED
RNA STAT-60TM: 100 ml or 200 ml bottle
containing a red solution of RNA STAT-60TM
PREPARATION
Ready to use.
STORAGE
Refrigerate at 2-8oC. Protect from exposure to light.
STABILITY
9 months. Refer to expiration date stamped on label.
4. REAGENTS REQUIRED, BUT NOT SUPPLIED
Chloroform (ACS grade)Isopropanol (ACS
grade)Ethanol (ACS grade)
5. PROTOCOL
RNA/mRNA isolation by the RNA STAT-60TM method includes the following
steps:
1. Homogenization
RNA STAT-60TM
(1
ml per 50-100 mg tissue, or 5-10 x 106 cells)
2. RNA Extraction
1 vol. of homogenate +0.2 vol. of chloroform
3. RNA Precipitation
0.5 vol. of isopropanol
4. RNA Wash
75% ethanol
Unless stated otherwise the procedure is carried
out at room temperature.
5.1 HOMOGENIZATION
A. TISSUES
Homogenize
tissues samples in the RNA STAT-60TM (1 ml/50-100mg tissue)
in a glass-Teflon or Polytron homogenizer. Sample volume should
not exceed
10% of the volume of the RNA STAT-60TM used for homogenization.
B. CELLS
Cells
grown in mono layer are lysed directly in a culture dish by adding
the RNA STAT-60TM (1 ml/3.5cm petri
dish) and passing the cell lysate several
times through a pipette. Cells grown in suspension are sedimented
then lysed
in the RNA STAT-60TM (1 ml per 5-10 x 106
cells) by repetitive pipetting.
Washing calls before addition of the RNA STAT-60TM
should
be avoided as this increases the possibility of mRNA degradation.
5.2 RNA EXTRACTION
Following homogenization, store the
homogenate for 5 min at room temp to permit
the complete dissociation of nucleoprotein complexes. Next, add
0.2 ml of chloroform per 1 ml of
the RNA STAT-60TM , cover the sample tightly,
shake vigorously for 15 seconds and let
it stay at room temperature for 2-3 minutes.
Centrifuge the homogenate at 12,000g (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.
RNA remains exclusively in the aqueous phase
whereas DNA and proteins are in the interferes
and organic phase. The volume of the aqueous phase is about 60%
of the volume of RNA STAT-60TM
used
for homogenization.
5.3 RNA PRECIPITATION
Transfer the aqueous phase to a fresh
tube and mix with isopropanol. Add 0.5 ml of
isopropanol per 1 ml of the RNA STAT-60TM
used
for homogenization. Store samples at room temp for 5-10 minutes
and, centrifuge at 12,000g (max.) for
10 min at 4oC. RNA precipitate (often visible before
centrifugation) forms a white pellet at the bottom of the tube.
5.4 RNA WASH
Remove supernatant and wash the RNA
pellet once with 75% ethanol by vortexing
and subsequent centrifugation at 7,500g (max.) for 5 min
at 4oC. Add at least 1 ml
of 75% ethanol per 1 ml of the RNA STAT-60TM
used
for the initial homogenization.
At the end of the procedure, dry the RNA pellet
briefly by air-drying or in a vacuum (5-10
min.). It is important not to let the RNA pellet dry completly
as it will greatly decrease its solubility.
Do not use the Speed-Vac for drying. Dissolve the
RNA pellet in water or in 1 mm EDTA, pH 7, or 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 may be required to dissolve RNA samples. Diethylpyrocarbonate
(DEPC) treated RNase-free solutions1 should be used for
solubilization of RNA.
6. EXPECTED YIELD AND PURITY
Expected yield of total RNA:
a.) Tissues (ug/mg tissue): liver,
spleen, 7-10 ug; kidney, 3-4 ug; skeletal muscles,
brain, 1-1.5 ug; placenta, 1-4 ug.
b.) Cultured cells (ug/10 6
cells): epithelial cells, 10-15 ug, fibroblasts, 5-7 ug. The
final preparation of total RNA is free of DNA and proteins and has
a 260/280
ratio > 1.8.
7. NOTES AND COMMENTS
1. For isolation of RNA from a small
amount of cells or tissue (1-10mg): homogenize
samples in 0.8 ml of the RNA STAT-60TM, transfer the homogenate
to the eppendorf tube and follow the isolation protocol with the
exception of the
RNA precipitation which should be carried out for 30 min at 4
oC.
2. Following homogenization (before addition
of chloroform) samples can be stored
at -70oC for at least 2 weeks.
3. An additional precipitation may be
necessary to use RNA isolated by the RNA
STAT-60TM in enzymatic assays.
Following solubilization, precipitate RNA
in the presence of 0.2 M NaCl with two volumes of
ethanol for 15 minutes
at 4oC. The PCR and RNase protection assays do
not require this traditional
precipitation step.
4. Hands and dust may be the major source
of the RNase contamination. Use gloves
and keep tubes closed. The use of sterile, disposable polypropylene
tubes is recommended
throughout the procedure.
8. SPECIAL HANDLING AND PRECAUTIONS
The RNA STAT-60TM contains poison (phenol)
and irritant (guanidinium thiocyanate).
CAN BE FATAL. When working with the RNA STAT-60TM
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.
9. 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, 156-189.
3. Kedzierski, W. and John Porter. 1991.
A Novel Non-enzymatic Procedure for
Removing DNA Template from RNA Transcription Mixtures. BioTechniques
10:210-214.
RNA
STAT-60TM is a trademark of Tel-Test
Inc.
PCR is subject of patents granted to Cetus
Corporation.
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