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1. INTRODUCTION
RNA STAT-30TM kit (patient pending) is a new and innovative
total RNA isolation/mRNA enrichment kit
that is based on the popular guanidinium
phenol "single-step method" of
total RNA isolation. The RNA STAT-30TM kit utilizes an
extraction reagent containing phenol, guanidinium, and proprietary
stabilizers and combines the dependable
"single-step method" with the speed and selectivity
of push affinity column chromatography .
The RNA STAT-30TM kit isolates total
RNA and enriches for mRNA from tissues and
cells of human, animal, plant, yeast, bacterial and viral
origin. The RNA is isolated within 3 minutes with minimal low-speed
(2,000g) centrifugation and no further purification
is required for many subsequent procedures including
Northern blotting, molecular cloning, RNase protection or PCR. The
RNA STAT-30 TM
kit utilizes a proprietary coated resin that effectively absorbs
RNA while allowing contaminants such as
guanidinium, phenol, detergents, salts, polysaccharides and
glycogen to wash through a push column. The RNA STAT-30 TM
kit quickly and effectively eliminates contaminants
from RNA preps that can cause high background levels,
lead to inaccurate quantitation of RNA, inhibit enzymes, or interfere
with sample activity and consistency2
. The RNA STAT-30TM total RNA/mRNA kit consistently provides
high quality undegraded RNA preps offering:
*Minimal low speed (200g)
centrifugation required
*Greater RNA prep consistency
*Extracts 30-150% more total
RNA/mRNA than any other method
*Northern blot/PCR-ready
RNA in under 30 minutes
*Direct RNA binding to anion
exchange resin
*Purer RNA/mRNA free of
contaminating phenol, detergents, salts, glycogen,
proteins, polysaccharides and other non-RNA contaminants
*Isolates RNA from 5 grams
of tissue or 5 x 106 cells
*Enzymatic assay ready RNA
in 30 minutes
2. KIT COMPONENTS SIZE
STORAGE
TL
2001 Solution 1 Extraction Reagent 50 ml 2-8oC
TL 2002 Solution 2 Washing Reagent
60 ml 2-8oC
TL 2003 Solution 3 Equilibration Reagent
60 ml 2-8oC
TL 2004 Solution 4 Binding Reagent
30 ml 2-8oC
TL 2005 Solution 5 Elution Reagent
60 ml 2-8oC
TL 2006 Solution 6 DEPC Ultra pure
Water 60 ml 2-8oC
TL 2007 RNA STAT-30 Affinity push columns
50 ea 2-8oC
TL 2008 RNA STAT-30 Collection Tubes 50 ea
R.T.
TL 2009 RNA STAT-30 3 cc Syringes
ITEMS REQUIRED BUT NOT SUPPLIED
Chloroform (ACS grade) and Ethanol
(ACS grade)
3. METHOD
Total RNA/mRNA isolation by the RNA
STAT-30TM method includes the following
steps:
1. Homogenization RNA STAT-30
TM
extraction
reagent (1 ml per 100 mg of tissue, or 10 x 106 cells).
2. RNA Extraction 1 vol. homogenate
+ 0.2 vol. of chloroform.
3. Column Washing Add 1 ml of
washing reagent to column.
4. Column Equilibration Add 1
ml of equilibration reagent to column.
5. RNA Immobilization Add 0.25
volume of column binding reagent to aqueous phase,
mix, and apply to column.
6. RNA Wash Wash immobilized
RNA with 75% ethanol.
7. RNA Elution Elute RNA from
column with 200 ul of elution buffer or DEPC water.
Unless stated otherwise the procedure is
carried out at room temperature.
3.1 HOMOGENIZATION
(a.) Homogenize tissue samples with
Solution 1 (RNA STAT-30TM extraction reagent)
(1 ml per 100 mg tissue with a few strokes in a glass-Teflon
homogenizer).
Sample
volume should not exceed 10% of the volume of the extraction reagent
used for homogenization.
(b.) To isolate RNA from cells grown
in suspension, sediment cells and lyse them by addition
of 1 ml Solution 1 (RNA STAT-30TM extraction reagent) per
10 x 106
cells. Cells grown in mono layer are lysed directly in the
culture dish by the addition
of Solution 1 (RNA STAT-30TM extraction reagent) (1
ml per 3.5 cm petri
dish or 102 (ex T-30 flask = 30 cm2 = 3 ml).
Solubilize the RNA by passing
the lysate a few times through a pipette.
3.2 RNA EXTRACTION
Following homogenization, store homogenate
for 5 minutes at room temperature. Add
0.2 ml of chloroform per 1 ml of the RNA STAT-30TM extraction reagent, cover
the sample tightly, shake vigorously for 15 seconds and let
it stay at room temperature for 2-3 minutes.
Centrifuge the homogenate at (2,000g 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 inter phase
and organic phase. The volume of the aqueous phase is about 60%
of the volume of RNA STAT-30 TM extraction reagent used
for homogenization.
3.3 COLUMN WASHING
Note: Use one column per 50-100 mg tissue
or 5-106 cells (1-2 ml extraction reagent).
1. Remove syringe plunger from
syringe barrel and attach syringe to the column luer lock.
2. Wash each column with 1 ml of
Solution 2 (washing reagent). Fill the syringe with 1
ml of Solution 2 (washing reagent) and eject reagent through
column by depressing
syringe plunger. Discard flow-through.
3.4 COLUMN EQUILIBRATION
Equilibrate each column with 1 ml
of Solution 3 (Equilibration reagent). Fill the syringe with
1 ml of Solution 3 (Equilibration reagent) and eject reagent
through column by depressing syringe plunger.
Discard flow-through.
3.5 RNA IMMOBILIZATION
1. Transfer the aqueous phase containing
RNA to a fresh tube and add 0.25 volume of
Solution 4 (binding reagent). Gently mix with pipet and add
two volumes (original
aqueous phase) of 100% ethanol. Store at room temperature
for 5 minutes.
2. Remove syringe plunger from syringe
barrel and attach syringe barrel to the column luer
lock.
3. Add RNA sample slowly (1 drop/second)
through column by depressing the syringe
plunger. Discard flow-through.
Note: The RNA sample flow-through can
be saved to later verify column binding
efficiency.
The RNA is now bound and immobilized
on the column.
3.6 RNA WASH
1. Remove syringe plunger from syringe
barrel and attach syringe barrel to the column luer
lock.
2. Wash column bound RNA with 1 ml of
75% ethanol. Eject ethanol wash solution through
column by depressing the syringe plunger. Discard flow-through.3.
Dry the immobilized
RNA briefly by air drying for 5-10 minutes or blowing air
through the column
with a syringe.
3.7 RNA ELUTION
1. Transfer column to RNase free 1.5
ml collection tube (provided).
2. Elute RNA off column slowly (1 drop/second)
with 200 ul of Solution 5 (elution reagent).
(See Section 4.6) Eject elution reagent through the column by depressing
the syringe plunger
and collect RNA. Repeat a second time using a fresh RNase free
1.5 ml microfuge tube.
3. Measure 260-280 ratios of each 200 ul
fraction and combine as necessary to maximize
yield.
4. NOTES AND COMMENTS
1. For isolation of RNA from small amounts
of cells or tissues (1-10 mg); homogenize samples
in no less than 0.8 ml of the RNA STAT-30TM
extraction
reagent, transfer the homogenate(s) to 1.5 ml microfuge tube(s), and
add 160 ul of chloroform
and follow isolation protocol.
2. Following homogenization (before addition
of chloroform) samples can be stored at --70oC
for 3 months.
3. Hands and dust may be the major source
of RNase contamination.
4. The immobilized RNA can be eluted with
most any appropriate RNase free buffer required
for subsequent procedures.
5. To concentrate the RNA, pass original ((100
ul-200 ul) fraction through column 3 times.
6. Solution 6 (DEPC water) may be substituted
for Solution 5 (elution buffer).7. The mini
columns can achieve RNA recovery rates of up to 99%.
5. SPECIAL HANDLING PRECAUTIONS
The extraction reagent contains poison
(phenol) and irritant (guanidinium thiocyanate). CAN
BE FATAL. When working with the extraction reagent use gloves
and eye protection (shield, safety goggles).
Do not get on skin or clothing. Avoid breathing vapor.
Read 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.
6. REFERENCES
1. Chomcyzynski P. and Sacchi N. (1987)
Single-Step method of RNA isolation by acid
guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem.
162, 156-159.
2. Puissant, C. and L. Houdebine. 1991.
An Improvement of the Single-Step method of
RNA Isolation by Acid Guanidinium Thiocyanate-Phenol-Chloroform
extraction. Biotechniques
8:148-149.
3. Sambrook J., Fritsch E.F. and Maniatis
T. (1989) Molecular Cloning. Cold Spring harbor
Laboratory, Cold Spring Harbor, N.Y.
4. 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, RNA STAT-60, and RNA STAT-30
are trademarks of Tel-Test "B", Inc.
CAT. NO. TL 2000 KIT
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