*Isolates high quality RNA/DNA from same sample in
*1ml isolates RNA/DNA from 100 mg tissue or 10 x 106 cells
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.
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.)
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
RNA/mRNA isolation by the RNA-DNA STAT-60 method includes the
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
3. RNA Precipitation 0.5 vol. of isopropanol.
4. RNA Wash 75% ethanol. Unless stated otherwise the
procedure is carried out at room temperature.
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
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
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
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 >
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
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
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.
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.
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.