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.


Phase separation

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 12,000g 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.


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 15-30oC for 10 min

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


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 2-8oC.


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.