Protocol of RNA Isolation From Tissue
Using TRIZOL
Updated 2001-10-23/ Mikael Altun
-
Add 1 ml TRIZOL reagent/50-100mg tissue and
homogenize.
-
Incubate the homogenized sample for 5 minutes at
room temperature. Add 0.2ml of chloroform per 1ml of TRIZOL
reagent. Cap sample tube securely. Shake tube vigorously by hand for 15
seconds (option vortex 10 seconds) and incubate it at room temperature
for 2-3 minutes. Transfer the homogenate into several 1.5-ml eppendorf
tubes.
-
Centrifuge the sample at 11,400 rpm (about 11,500xg
for IEC 851 Rotor) for 15 minutes at 2-8°C. Transfer the upper aqueous
phase (containing the RNA) to fresh tubes.
-
Precipitate the RNA by adding 1ml (equal volume
of aqueous phase) of 100% isopropanol shake tubes by hand and vortex
for 10 seconds. Incubate sample at room temperature for 10 minutes and
centrifuge at 11,400 rpm (about 11,500xg for IEC 851 Rotor) for 10 minutes
at 2-8°C and discard the supernatant.
-
Wash the RNA pellet twice with 75% ethanol
(DEPC-water diluted), adding at least 1ml of 75% ethanol per 1ml
of TRIZOL reagent used for the initial homogenization. Mix the samples
by vortexing and centrifuge at 11,400 rpm (about 11,500xg for IEC 851 Rotor)
for 5 minutes at 2-8°C.
-
Dry the RNA pellet in vacuum or air-dry for 5-10
minutes. Dissolve RNA pellet in some DEPC-treated water by passing
the water a few times trough a pipette tip and incubating for 10 minutes
at 55 to 60°C. Store samples frozen at -70°C.
-
Quantify RNA by diluting 1ml
in 99ml
ddH2O and read sample in eppendorf BioPhotometer using the RNA
program.
Alternative quantify RNA by diluting 1or 2 or 5ml
in 700ml
ddH2O and read the A260 and A280. (generally
>1.80)
1A260=40mg/ml
(RNA)
RNA (mg/ml)
= A260 x 40 x diluted times