66
HANS GERD NOTHWANG AND FRIEDHELM HILDEBRANDT
Preparation
of total RNA
Gomponent
Stock
Final
Amount
Goncentration
Goncentration
a) guanidinium thiocyanate
4M
47.2 g
b) sodium citrate (pH 7.0)
1M
25mM
1.25 ml
c) sodium lauryl sarkosinate 10%
O.So/o
2.5 ml
d) H20 (DEPG-treated)
to SO ml
e) ß-mercaptoethanolb
14.3 M
0.1 M
0.35 rnl
a Mix reagents a-d at 65°C. This stock solution can be stored at least 3 months at room
temperature.
b Add just before use.
2a) For tissue, mince 100 mg freshly prepared tissue and homogenize with
1ml of solution D in a glass-Teflon homogenizer.
2b) For 10 7 cultered cells, either spin suspension cells and discard supernatant, or remove the culture medium from cells grown in monolayer
cultures. Add 1ml solution D and pass lysate through a pipet 10 times.
Note: Carry out all steps at room temperature unless otherwise stated.
3. Transfer cells into a 4 ml polypropylene tube. Add 0.1 ml of2 M sodium
acetate (pH 4.0) and mix. Add 1ml water-saturated phenol, mix, and
add 0.2 ml of chloroform:isoamyl alcohol (49:1). Shake vigoursly
and cool on ice for 15 min.
4. Spin 20 min at 10000 g (9000 rpm in a SS-34 rotor), 4°C.
5. Transfer upper aqueous phase to a fresh tube. Precipitate RNA with 2.5
ml (2.5 vol) ice-cold 100o/o ethanol, place >30 min at -20°.
6. Spin 10 min at 10000 g, 4°C.
Note: For isolation ofRNA from tissues with a high glycogen content (e.g.
liver), wash out glycogen from RNA pellet byvortexing in 4 M LiCl after the
ethanol precipitation. Sediment insoluble RNA 10 min at 5000 g and follow
reminder of protocol.
7. Dissolve RNA in 0.3 ml solution D and transfer to a 1.5 ml microcentrifuge tube. Precipitate RNA with 0.75 ml ice-cold 100o/o ethanol
>30min at -20°C.
8. Spin 10 min at 10000 rpm, 4°C.
HANS GERD NOTHWANG AND FRIEDHELM HILDEBRANDT
Preparation
of total RNA
Gomponent
Stock
Final
Amount
Goncentration
Goncentration
a) guanidinium thiocyanate
4M
47.2 g
b) sodium citrate (pH 7.0)
1M
25mM
1.25 ml
c) sodium lauryl sarkosinate 10%
O.So/o
2.5 ml
d) H20 (DEPG-treated)
to SO ml
e) ß-mercaptoethanolb
14.3 M
0.1 M
0.35 rnl
a Mix reagents a-d at 65°C. This stock solution can be stored at least 3 months at room
temperature.
b Add just before use.
2a) For tissue, mince 100 mg freshly prepared tissue and homogenize with
1ml of solution D in a glass-Teflon homogenizer.
2b) For 10 7 cultered cells, either spin suspension cells and discard supernatant, or remove the culture medium from cells grown in monolayer
cultures. Add 1ml solution D and pass lysate through a pipet 10 times.
Note: Carry out all steps at room temperature unless otherwise stated.
3. Transfer cells into a 4 ml polypropylene tube. Add 0.1 ml of2 M sodium
acetate (pH 4.0) and mix. Add 1ml water-saturated phenol, mix, and
add 0.2 ml of chloroform:isoamyl alcohol (49:1). Shake vigoursly
and cool on ice for 15 min.
4. Spin 20 min at 10000 g (9000 rpm in a SS-34 rotor), 4°C.
5. Transfer upper aqueous phase to a fresh tube. Precipitate RNA with 2.5
ml (2.5 vol) ice-cold 100o/o ethanol, place >30 min at -20°.
6. Spin 10 min at 10000 g, 4°C.
Note: For isolation ofRNA from tissues with a high glycogen content (e.g.
liver), wash out glycogen from RNA pellet byvortexing in 4 M LiCl after the
ethanol precipitation. Sediment insoluble RNA 10 min at 5000 g and follow
reminder of protocol.
7. Dissolve RNA in 0.3 ml solution D and transfer to a 1.5 ml microcentrifuge tube. Precipitate RNA with 0.75 ml ice-cold 100o/o ethanol
>30min at -20°C.
8. Spin 10 min at 10000 rpm, 4°C.