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INTRODUCTION
The entire procedure for RNA/DNA isolation
using RNA-DNA STAT-60 can be completed in less than 90 minutes.
These are the most effective methods for isolation of RNA and DNA
isolation from the same sample. The recovery of undegraded RNA and
DNA is 30-150% greater than with any other method of RNA/DNA
isolation.
APPLICATION
The total
RNA/DNA isolated by RNA-DNA STAT-60 is used for Northern and
Southern analysis, dot-blot hybridization, poly A+ selection, in
vitro translation, RNase protection assay, molecular cloning, and
polymerase chain reaction from human, animal, plant without any
additional RNase/DNase treatment. Multi-sample processing is a great
advantage of using these simple single reagents. Excellent recovery
of RNA/DNA permits the use of this product for isolation of RNA/DNA
from very small biological samples (biopsies etc.)
REAGENTS SUPPLIED
RNA STAT-60 and DNA
STAT-60Preparation: Ready to use.Storage: Refrigerate at
2-8oC.Stability: Refer to expiration date stamped on
label.
REAGENTS REQUIRED BUT NOT SUPPLIED
Chloroform (ACS
grade), Isopropanol (ACS grade), and Ethanol (ACS grade).
PROTOCOL
RNA/mRNA isolation by the RNA-DNA STAT-60
method includes the following steps:
1. Homogenization RNA
STAT-60 (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: Homogenized tissue
samples in the RNA STAT-60 (1 ml/ 50-100 mg tissue) in a
glass-Teflon or Polytron homogenizer. Sample volume should of the
RNA STAT-60 used for homogenization.
B. Cells: Cells grown
in mono layer are lysed directly in a culture dish by adding the RNA
STAT-60 TM
(1 ml per 5-10 x
106 cells) by repetitive pipetting. Washing cells
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 minutes at room temperature 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. Centrifugate 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. RNA remains exclusively
in the 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 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 temperature for 5-10 minutes
and centrifuge at 12,000 g (max.) for 10 minutes 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 and subsequent centrifugation at 7,500 g
(max.) for 5 minutes 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 completely as it will greatly decrease
its solubility. Do not use the SpeedVac 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 55-60 oC may be required to dissolve
RNA samples. Diethylpyrocarbonate (DEPC) treated RNase-free
solutions 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/106 cells): epithelial cells, 10-15 ug,
fibrolasts, 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 (1-10 mg): 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 minutes 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. Hand 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 PRECAUTIONS
The RNA
STAT-60TM contains poison
(phenol) and irritant (guanidinium thiocyante). 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.
PROTOCOL FOR DNA EXTRACTION FROM SAMPLE USED FOR RNA
EXTRACTION
DNA REVERSE EXTRACTION
Remove aqueous layer containing
RNA. Add 800 ul of DNA STAT-60 reagent per 1 ml of RNAzol, RNAzol B
or RNA STAT-60 used for the initial homogenization. Add 0.2 ml of
chloroform per 1 ml of DNA STAT-60, shake vigorously for 15 seconds
and let it stay at room temperature for 2-3 minutes. Centrifuge the
homogenate at 2000 g (min.) - 12,000 g (max.) for 15 minutes at
4oC. Following centrifugation the homogenate separates
into two phases: a lower organic phase and the upper aqueous phase.
DNA remains in the aqueous phase whereas RNA and proteins are in the
inter phase and organic phase.
DNA PRECIPITATION
Transfer the aqueous phase to a fresh
tube and mix with isopropanol. Add 0.5 ml of isopropanol per 1 ml of
the DNA STAT-60 used for homogenization store samples at room
temperature for 5-10 minutes and centrifuge at 12,000 g (max.) for
10 minutes at 4oC, The DNA precipitate forms a small
clear to white pellet at the bottom of the tube.
DNA WASH
Remove supernatant and wash the DNA pellet
once 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 1 ml of the DNA STAT-60 used for the initial
homogenization.At the end of the procedure, dry the DNA pellet
briefly by air drying or in a vacuum (5-10 min.). Dissolve the DNA
pellet in water or in 1 mm EDTA, pH 7. 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 DNA samples.
SPECIAL HANDLING PRECAUTIONS
The DNA
STAT-60TM contains an irritant
(guanidinium salts). Can be fatal. When working with DNA STAT-60 use
gloves and eye protection (shield, safety goggles). Do not get on
skin or clothing. Avoid breathing vapor. Read warning note on
bottle.
REFERENCES
1. Chomczynski, P. and Saachi, 1987, Single
Step Method of RNA Isolation by Acid Guanidinium
Thiocyanate-Phenol-Chloroform Extraction. Biotechniques 8:148-149.
Additional references available upon request.
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