RNA STAT 50 LS
*Extraction reagent for
*Effective method of RNA isolation from biological fluids (blood,
plasma, amniotic fluid and CSF).
*Excellent for polymerase chain reaction without the hassle of
additional DNase purification.
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.
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
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.
Reagent supplied: RNA STAT-50TM
LS. Reagents required, but not supplied: chloroform, isopropanol and
The methods include the following steps:
1. Homogenization 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
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
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
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
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
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
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
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
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, 156-159.
3. Chomzynski P. (1989) The RNAzolTM