RNA Extraction from tissue using Phenol-Chloroform Extraction
- TRIzol Reagent
Set up requirements
Fumehood, vortexer, micropipette, chilled micro-centrifuge, pellet pestle homogenizer, and all standard equipment of molecular biology laboratory.
- Always use disposable nitrile gloves
- Use sterilize tips or RNAase/DNAase free filter tips
- Use disposable sterile plastic ware or RNAase free glassware
- Before starting the extraction, clean the workstation with surface decontamination that destroys RNAases (RNA-zap).
- Chill the micro-centrifuge to 4∞C
- Add 1ml of TRIzol per 100 mg of fresh tissue or tissue stored in RNA later and mince on ice using sterile scalpels. The tissue is further homogenized with sterile pellet pestle probe.
* Frozen tissue should be thawed at room temp and removed from RNA later before adding TRIzol reagent.
- Transfer the tissue lysate to 2ml sterile micro-centrifuge tube
- For complete dissociation of nucleoprotein complexes, let the homogenate settle at room temp for 5 min.
- Add 200μl of chilled Chloroform and vortex vigorously for 20 sec.
*Thorough mixing and shaking is critical for phase separation.
- Let the homogenate to settle at room temp for 5 min.
- Centrifuge the homogenate at 12,000 x g for 20 min at 4∞C.
- Carefully transfer the upper aqueous phase (600μl) over 500 μl of chilled isopropanol in micro-centrifuge tube.
*Store interphase for further isolation of DNA.
- Let the RNA, to precipitate in isopropanol for 1 to 2 hr at -20∞C.
- After precipitation, centrifuge the RNA sample in isopropanol 15,000 x g for 20 min at 4∞C.
- Carefully extract the RNA precipitated in the form of gel at the bottom the the micro-centrifuge.
- RNA pellet so obtained is washed with 200μl of 75% ethanol proceeded with brief vortexing and
centrifugation at 7500 g for 10 min at 4°C.
- Air-dried the washed RNA pellet for 5 min
- Using RNAase free water re-suspend the air-dried RNA pellet.
Now, you can proceed with RNA quantity and quality check and followed by its conversion into cDNA.