Updated 2001-10-23/ Mikael Altun
1. Add 1 ml TRIZOL reagent/50-100mg tissue and homogenize.
2. 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.
3. 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.
4. 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.
5. 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.
6. Dry
the RNA pellet in vacuum or air-dry for 5-10 minutes. Dissolve RNA pellet in
some (sugg vol 100µl see DNAse protocol) DEPC-treated water by passing
the water a few times trough a pipette tip. Ensure that pellet is dissolved.
7. DNAse
treat RNA samples using DNAse-I (Ambion)
Add 2µl (4 units) per 100µl RNA solution + 10µl
10XDNAse-I buffer
Incubate at 37°C for 25min
Inactivate by addition of 0.1 volume (ca 10µl)
DNAse inactivation reagent (slurry)
Incubate 2min at RT with occasional mixing
Spin at 10 000g at RT for 1.5min
Transfer supernatant to fresh tubes for
storage.
8. Quantify RNA by diluting 3µl in 97µl ddH2O and read sample in eppendorf BioPhotometer using the RNA program. Store samples frozen at -70°C.
1A260=40µg/ml (RNA)
RNA (mg/ml) = A260 x 40 x diluted times