RNA extraction using the 'home-made' Trizol substitute
Prepare the components for the home-made Trizol substitute. 
Mix the components and stir at room temperature until completely dissolved (30-60 minutes).
Do not heat the solution.
Store at 4 °C in a glass bottle protected from light.
Add 5 volumes of the home-made Trizol substitute to the sample.
When working in 1.5 mL tubes, for practical reasons, the minimal and maximal volume of the sample is 30 and 200 µl, respectively.
Vortex vigorously for 10-30 seconds (depending on the viscosity and protein and nucleic acid concentration of the sample).
Incubate at room temperature for 5 minutes.
Add 1 volume of chloroform (relative to the original volume of the sample).
Mix vigorously 10-20 times by inverting the tube.
Do not vortex to avoid breaking DNA.
Incubate at room temperature for 5 minutes.
Spin for 10-15 minutes at >12,000 g and 4 °C (depending on the viscosity and protein and nucleic acid concentration of the sample).
Transfer the upper aqueous phase into a new tube.
Avoid the white DNA precipitate at the interphase.
If the sample volume was 100 µl, the aqueous phase should be ~450 µl.
Add 1.1 volumes of isopropanol (relative to the volume of the aqueous phase).
If low RNA content is expected in the sample, prior to isopropanol, add >10 µg of glycogen to facilitate precipitating nucleic acids and spotting the pellet.
Recommended final concentration of glycogen is ~50–150 µg/ml.
Mix well by inverting the tube.
Incubate 30-60 minutes at 4 °C or at room temperature.
Spin for 30-45 minutes at >12,000 g and 4 °C.
Discard the supernatant.
Add 1 volume of 70% (aq.) ethanol (relative to the volume of the aqueous phase).
Mix gently.
Ensure that the pellet is submerged in the solution and not sticking to the tube wall.
Incubate 10 minutes at room temperature.
Spin for 10 minutes at >12,000 g and 4 °C.
Discard the supernatant.
Let the pellet dry at room temperature, so that no traces of 70% ethanol remain.
Drying under vacuum is not recommended because of overdrying that makes it harder to dissolve the pellet.
Completely dissolve the pellet in RNase-free water.
