8.3.2. Protocol 2 – TRIzol extraction and isopropanol precipitation
This protocol uses TRIzol®; a proprietary mixture of phenol and high concentration salt solution (InVitrogen). The RNA is recovered through isopropanol precipitation.
1. Homogenize bees directly at 4oC in
TRIzol® reagent in a glass-walled blender or mortar and pestle. Use
1 ml reagent per bee (~120 mg tissue).
2. Add 0.5 ml chloroform per bee.
3. Shake hard for 1 minute.
4. Centrifuge 8,000 g; 15 minutes; 4oC.
5. Recover the upper (aqueous) layer containing the nucleic acids, discarding the lower red (organic) phase and the semi-solid, white interphase (containing proteins and lipids).
6. Add an equal volume of iso-propanol, mix and precipitate at -20oC for at least 15 minutes.
7. Centrifuge 8,000 g; 15 minutes; 4oC.
9. Remove iso-propanol supernatant.
10. Resuspend nucleic acid pellets in 100 µl RNase-free water.
11. Determine nucleic acid concentration and purity (see section 8.4.; “Nucleic acid quality assessment”).
12. Store as two separate 50 μl aliquots at -80oC, one for working with and one for storage.