Total RNA Isolation Using TRIzol:

Prepare Tadpole brain tissue

Dissect Tadpole brain tissue from stage 45-48 tadpole ( 50 or more tadpole brains may be used in order to get enough tissue ) and put into 1.5 ml eppendorf tube in a proper size of beaker containing dry ice and ethanol sitting on dry ice.

Homogenization

Homogenize tissue samples in 1 ml of TRIzol reagent per 50-100 mg of tissue using a glass-homogenizer. Homogenization process should be finished as quick as possible to avoid RNA degredation. The sample volume should not exceed 10% of the volume of TRIzol reagent used.

Phase separation

Incubate the homogenized samples for 5 min at 15 to 30^oC.

Add 0.2 ml chloroform per ml of TRIzol added

Shake tubes vigorously by hand for 15 seconds

Incubate at 15-30^oC for 2-3 min

Centrifuge the sample at ≤12,000g for 15 min at 2-8^oC

RNA remains exclusively in the upper aqueous phase. The volume of aqueous phase is about 60% of the volume of TRIzol used for homogenization.

RNA precipitation

Transfer the aqueous phase to a fresh tube

Add 0.5 ml isopropyl alcohol per 1 ml TRIzol added, vortex

Incubate sample at 15-30^oC for 10 min

Centrifuge the sample at ≤ 12,000g for 10 min at 2-8^oC

RNA wash

Remove supernatant.

Wash RNA pellet once with 75% ethnol, adding at least l ml of ethnol per ml TRIzol used, vortex.

Centrifuge at ≤7,500g for 5 min at 2-8^oC.

Redissolving RNA

Air dry RNA pellet 5-10 min

Dissolve RNA in RNase-free water and incubating for 10 mins at 55 to 60 ^oC.

Dilute RNA sample 50 times with water and measure OD at 260/280.