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1. PRODUCT DESCRIPTION
The
efficient isolation of high quality undegraded poly (A+) mRNA is
essential for the study of eukaryotic genes. Since poly (A+)
mRNA comprises only 2-3% of total RNA, the total RNA extraction
method of choice must be efficient. The mRNA STAT-30 kit
extraction reagent extracts 30-150% more mRNA than any other method
making it especially suitable for poly (A+) isolation. The
mRNA STAT-30 isolation kit combines state of the art RNA isolation
technology with the speed and selectivity of oligo(dT) cellulose
push column affinity chromatography offering:
*Reproducible
yields of undegraded poly (A+) mRNA in 30 minutes.
*Guanidinium
based ribonuclease protection system.
*Isolation of mRNA directly
from tissues and cells without intermediate total RNA
isolation.
*Exceptional quality poly (A+) mRNA that is suitable
for constructing DNA libraries, SI analysis, Northern hybridization
and dot-blot analysis.
*Twenty mini preps of (1-100 mg tissue) or
(103-107 cells).
2. KIT COMPONENTS
Note: All
mRNA STAT-30 kit components are treated with diethylpyrocarbonate
(DEPC) and are guaranteed to be RNase-free.
CAT. NO.
COMPONENTS
SIZE STORAGE
TL 3001
Solution 1 Extractant
Reagent
50
ml
2-8 oC
Tl
3002 Solution 2
Washing
Reagent
60
ml
2-8 oC
TL 3003
Solution 3 Ultrapure DEPC
Water
60
ml
2-8 oC
TL 3004
Solution 4 Binding Suffer
(1x)
60
ml
2-8 oC
TL 3005
Solution 5 Binding Buffer
(2x)
60
ml
2-8 oC
TL 3006
Solution 6 Elution
Buffer
60
ml
2-8 oC
TL 3007
Solution 7 Sodium
Acetate
2
ml
2-8 oC
TL 3008
Solution 8 Storafe
Buffer
60
ml
2-8 oC
TL 3009
Pre-packed oligo (dT)-cellulose
columns 20
ea
2-8 oC
TL 3010
Syringes
20
ea
RT
TL 3011 Collection
Resevoirs
20
ea
RT
ITEMS REQUIRED BUT NOT SUPPLIED
Chloroform (ACS
grade), Isopropanol (ACS grade), Ethanol (ACS grade), and sterile
1.5 microfuge tubes.
3. PROCEDURE
Note: Treat all
components in thid kit aseptically to avoid the introduction of
RNases. Gloves should be worn at all times during the
procedure.
Unless stated otherwise the procedure is carried out
at room temperature.
A.
HOMOGENIZATION
1. TISSUES: Homogenize tissue samples in Solution 1 (extraction
reagent) (1 ml/10 ug-100 mg tissue) in a glass-Teflon or
polytron homogenizer. Sample volume should not exceed 10%
of the volume of extraction reagent used for
homogenization.
2. CELLS: Cells grown in monolayer are lysed directly in a culture
dish by adding Solution 1 (extraction reagent) [1 ml/3.5 cm
petri dish or 10 cm2 (ex. T-30 Flask = 30 cm2
= 3 ml)] and passing the cell lysate several times through a
pipette. Cells grown in suspension are sedimented then lysed
in Solution 1 (extraction reagent) (1 ml per
103 - 107 cells) by repetitive
pipetting. Washing cells before addition of the extraction
reagent should be avoided as this increases the possibilty of mRNA
degradation.
B. RNA EXTRACTION
Following homogenization, store homogenate 5 minutes
at room tempture to permit complete dissication of nucleoprotein
complexes. Next,m add 0.2 ml chloroform per 1 ml
extraction reagent, cover sample tightly, shake vigorously (do not
vortex) for 15 secnds and let it stay at room temperature
2-3 minutes. Centrifugate 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 a colorless upper aqueous
phase. RNA remains exclusively in the aqueous phase whereas
DNA and proteins are in the interphase and organic phase. The
volume of the aqueous phase is about 60% of the volume of extraction
regeant used for homogeniztion. Transfer aqueous phase to a
fresh tube and store it at 4oC.
C. PREPARATION OF COLUMN(S)
Note: Use one column per (10-100 mg tissue) or
(103-107 cells).
1. Allow all kit
components to come to room tempture.
2. Wash each column
with 250 ul of Solution 2 (washing reagent). Attach
syringe to column leur lock fitting and remobe syringe
plunger. Fill syringe with 250 ul of Solution 2
(washing reagent) and eject reagent reagent through column by
depressing syringe plunger. Discard flow-through. Wash
cellulose 3 times with 1 ml of Solution 3 (ultrapure DEPC
water) as above.
3. Equilibrate olido (dT)-cellulose by
washing column(s) 3 times with 1 ml Solution 4
(binding buffer 1x). Fill syringe with 1 ml Solution 4
(binding buffer 1x) and eject buffer through column by depressing
syringe plunger. Store column(s) at room temperature with a
layer of Solution 4 (binding buffer 1x) over the
cellulose.
D. BINDING mRNA to OLIGO
(dT)-CELLULOSE
1. Carefully measure the
aqueous phase volume (from 3B) and add an equal volume of
Solution 5 (binding buffer 2x). Mix well and slowly
eject sample (1 drop/sec) through equilibrated oligo (dT)-cellulose
column by depressing syringe plunger. Discard
flow-through.
2. Wash column with 1 ml of Solution 4
(binding bufefr 1x). Discard flow-through. Repeat two
times.
E. ELUTION OF mRNA
1. Place column into collecton reservoir.
2.
Elute the poly (A+) mRNA by washing slowly with 3x 200 ul of
Solution 6 (elution buffer) warmed to 65oC.
Fill syringe with 200 ul of Dolution 6 pre-warmed (elution
buffer) and slowly eject buffer (1 drop/sec) into collection
reservoir. Repeat 2 times for a total of 3 washes with
Solution 6 (elution buffer). Store collection tube(s)
contaiing the poly (A+) mRNA on ice.
F.
PRECIPITATION OF POLY (A+) mRNA
To each tube
containing the poly (A+) mRNA flow-through add 0.1 volume of
Solution 7 (sodium acetate pH 5.2). Mix well and add 2.5
volumes of ice cold ethanol. Precipitate at -20C for at least
15 minutes. (A dry ice/alcohol bath is recommended).
Centrifuge sample for 15 minutes at 12,000g (4C). Remove
supernatant and wash RNA pellet once with 70% ethanol. Dry RNA
pellet under vacuum for 10-15 minutes and resuspend in the
appropriate buffer for use as specified in subsequent
procedures.
G. ISOLATION OF TOTAL RNA
1.
HOMOGENIZATION: Follow the procedure outlined in Section IIIA.
2.
RNA EXTRACTION: Follow the procedure outlined in Section IIIB.
3.
RNA PRECIPITATION: Transfer the aqueous phase to a fresh tube and
mix with isopropanol. Add 0.5 mL isoproponal per 1mL of the
extraction reagent used for homogenization. Store samples at
room temperature 5-10 minutes and centrifuge at 12,000g (max.) for
10 minutes at 4C. RNA precipitate (often visible before
centrifugation) forms a white pellet at bottom of tube.
4. RNA
WASH: Remove supernatant and wash the RNA pellet once with 75%
ethanol by vortexing and subsequent centrifugation at 7,500g (for 5
minutes at 4C. Add at least 1mL of 75% ethanol per 1mL of the
extraction reagent for the initial homogenization.
At the end of
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 Spee-Vac for drying. Dissolve RNA pellet in water or mM
EDTA, pH 7, or 0.5% SDS solution. Vortex or pass pellet a few
times through a pipette tip. Incubation for 10-15 minutes at
55-60C may be required to dissolve RNA samples.
Diethylpyrocarbonate (DEPC) treated RNase-free solutions should be
used for solubilization of RNA.
H. ISOLATION OF mRNA FROM TOTAL RNA (optional
protocol)
1. Measure total RNA solution volume
(from G4) and add an equal volume of Solution 5 (binding buffer
2x).
2. Follow procedure outlined in Sections III D, E and
F.
4. 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, 1-1.5ug; placenta 1-4ug.
B.
Cultured cells (ug/106 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.
5. COLUMN REGENERATION AND STORAGE:
A. After procedure, wash
column(s) with 1 mL of Solution 2 (washing reagent), discard
flow-through. Repeat two times.
B. Wash column(s) with 1mL
of Solution 8 (storage buffer). Discard flow-through.
Repeat two times. Store cellulose at 2-8C in Solution 8
(Storage buffer). The used cellulose can be stored in Solution
8 for a few days. For longer storage wash cellulose with 1mL
Solution 3 (ultrapure DEPC water). Discard flow-through.
Repeat two times. Finally wash cellulose with 1mL of ethanol,
vacuum dry, and store column(s) at -20C. The columns may be
re-sed several times.
6. NOTES AND COMMENTS:
A. For isolation of RNA from a
small amount of cells or tissue (1-10 mg); homogenize samples in 0.8
mL of extraction reagent, transfer homogenate to the eppendorf tube
and follow isolation protocol except RNA precipitation should
be carried out for 30 minutes at 4C.
B. Following
homogenization (before addition of chloroform) samples can be stored
at -70C for at least 3 months.
C. An additional
precipitation may be neccessary to use RNA isolated by the
extraction reagent in enzymatic assays. Following
solubilization, precipitate RNA in the presence of 0.2 M NaCl with
two volumes ethanol for 15 minutes at 4C. The PCR and Rnase
protection assays do not require this traditional precipitation
step.
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 also the warning
note on the bottle.
In case of contact immediately flush eyes or skin with large
amount of water for at least 15 minutes and seek immediate medical
attention.
References:
1. Aviv, H. & Leder, P. Purification of
biologically active globin messenger RNA by chromotography on
oligothymidylic acid-cellulose. Proc. Natl. Acad. Sci. USA
69:1408 (1972)
2. Chirgwin, J.M., Przybyla, A.E., MacDonald, R. J. & Rutter,
W.J. Isolation of biologically active ribonucleic acid from sources
enriched in ribonuclease.
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