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.
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.
Incubate the homogenized samples for 5 min at 15 to 30oC.
Add 0.2 ml chloroform per ml of TRIzol added
Shake tubes vigorously by hand for 15 seconds
Incubate at 15-30oC for 2-3 min
Centrifuge the sample at ²12,000g for 15 min at 2-8oC
RNA remains exclusively in the upper aqueous phase. The volume of aqueous phase is about 60% of the volume of TRIzol used for homogenization.
Transfer the aqueous phase to a fresh tube
Add 0.5 ml isopropyl alcohol per 1 ml TRIzol added, vortex
Incubate sample at 15-30oC for 10 min
Centrifuge the sample at ² 12,000g for 10 min at 2-8oC
Wash RNA pellet once with 75% ethnol, adding at least l ml of ethnol per ml TRIzol used, vortex.
Centrifuge at ²7,500g for 5 min at 2-8oC.
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.