* 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.
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:
The total RNA isolated by the RNA STAT-60TM
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
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
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
RNA/mRNA isolation by the RNA STAT-60TM method includes the
1. Homogenization RNA STAT-60TM (1 ml per 50-100 mg tissue,
or 5-10 x 10-6 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.
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.
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 sediment 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
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
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
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 30m min at 4 oC.
2. Following homogenization (before addition of chloroform)
samples can be stored at -70oC for at least 2
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
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
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.
1. Sambrook J., Fritsch E. F. and Maniatis T. (1989)
Molecular Cloning. Cold Spring Harbor Laboratory, Cold Spring
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.
is a trademark of Tel-Test Inc.
PCR is subject of patents granted to Cetus Corporation.