- Chloroform (without any additives, such as isoamyl alcohol)
- Isopropyl alcohol
- 75% Ethanol (in DEPC-treated water)
- RNase-free water or 0.5% SDS solution
The SDS solution must be prepared using DEPC-treated, autoclaved water. DEPC inactivates the RNases by the covalent modifications of the histidine residues.
Working area, gloves, pipettes etc. were wiped with RNase Blaster solution.
Growth medium on the cells was discarded and cells were washed with ice cold 1X PBS. The monolayer was then covered with 1 ml of l TRIzol and the cells were lysed and homogenized by repeated pipetting.
2. Phase Separation
- The homogenized samples were incubated for 5 minutes at 15 to 30°C for the complete dissociation of nucleoprotein complexes.
- 0.2 ml (200 microliters)of chloroform per 0.75 ml of TRIZOL LS Reagent was added. The tubes were shaked vigorously by hand for 15 seconds and incubated them at 15 to 30°C for 2 minutes.
- The samples were centrifuged for 15 minutes at no more than 12,000 g (4°C).
- The aqueous phase was transferred to other tubes. ( Following centrifugation, the mixture separates into a lower red, phenol-chloroform phase, an interphase, and a colorless upper aqueous phase. RNA remains only in the aqueous phase. The volume of the aqueous phase is about 70% of the volume of TRIZOL LS Reagent used for homogenization.)
3. RNA Precipitation
- The RNA was precipitated from the aqueous phase by mixing with 3 microlitre of glycogen and 500 microlitre of isopropyl alcohol.
- The mixture was centrifuged for 30 minutes at 12,000 × g (2 to 8°C).( The RNA precipitate forms a gel-like pellet on the side of the tube at bottom).
4. RNA Wash
- The supernatant was removed. The RNA pellet was washed once with 75% ethanol, adding 900 microlitre of 75% ethanol per 0.75 ml of TRIZOL LS Reagent used for the initial homogenization.
- The sample were inverted and mixed and centrifuged at 12,000 rpm for 30 minutes at 4 degree.
5. Redissolving RNA
- The RNA pellet was dried .
- RNA was dissolved in RNase-free water (or 0.5% SDS solution) by passing the solution through the pipette tip for a few times, and incubating for 10 minutes at 55 to 60°C.
Note: 0.5% SDS should not be used if RNA will undergo further enzymatic reaction. DNA can also be in 100% formamide (deionized) and stored at -70°C.
Precautions for preventing RNase contamination:
RNases can be introduced into the RNA preparation at any point accidently during the isolation procedure through improper technique. RNase activity is difficult to inhibit, so it is very essential to prevent its introduction. The following guidelines should be observed carefully while working with RNA. Always wear disposable gloves. Usually our skin contains many bacteria and molds that can contaminate the RNA preparation and be a source of RNases. Good and clean microbiological techniques will prevent microbial contamination. Use sterile, disposable plastic ware and automatic pipettes reserved for RNA work to prevent cross-contamination with RNases from shared equipment.