ISOLATION OF RNA FROM

ANIMAL SOURCE(TISSUE)
AIM:To isolate RNA from animal source using Trizol method.
Principle for RNA isolation:
Total RNA is isolated and separated from DNA and protein after
extraction with a
solution called as Trizol. Trizol is an acidic solution containing
guanidinium thiocyanate (GITC),
phenol and chloroform. GITC irreversibly denatures proteins
and RNases. This is followed by
centrifugation. Under acidic conditions, total RNA remains in
the upper aqueous phase, while
most of DNA and proteins remain either in the interphase or in
the lower organic phase. Total
RNA is then recovered by precipitation with isopropanol. RNase
enzymes can be inactivated by
including diethyl pyrrocarbonate (DEPC).
 PRINCIPLE FOR AGAROSE GEL
ELECTROPHORESIS:

Agarose gel electrophoresis is the easiest way of separating and analyzing nucleic acids
based on their charge by applying an electric field to the electrophoretic apparatus
where shorter molecules migrate more easily and move faster than longer molecules
through the pores of the agarose gel. Agarose, obtained from red algae makes an inert
matrix for the separation and most agarose gel for electrophoresis are made between
0.7% to 2% of agarose. The nucleic acids can be visualized in the gel by addition of
ethidium bromide that act as an intercalating agent for the nucleic acid bases. The speed
of migration of nucleic acids on the gel is affected by the given voltage, ionic strength
of the buffers.
 Materials and reagents required for Agarose gel
electrophoresis:

Nucleic acids and oligonucleotides, centrifuge machine, magnetic stirrer Casting tray ,Well
combs, Voltage source, Gel box, UV light source, Microwave.
Buffers and Solutions:
Agarose solutions, Electrophoresis buffer, RNA staining solution and Gel-loading buffer
Agarose solution: Measure 1 g of agarose powder with 100 mL 1xTAE in a microwavable flask
microwave for 1-3 min until the agarose is completely dissolved.
RNA Staining Solution: Ethidium bromide (10 mg/ml).
Ethidium Bromide: Add 1 g of ethidium bromide to 100 ml of H2O. Stir until the dye has dissolved.
Wrap the container in aluminum foil or transfer the %1 (10 mg/ml) solution to a dark bottle and
store at room temperature.
Electrophoresis Buffer; TAE, TPE and TBE
Gel-loading Buffer :
0.25% (w/v) bromophenol blue
0.25% (w/v) xylene cyanol FF
40% (w/v) sucrose in H2O
 Materials and reagents for trizol method:

Chloroform
Isopropanol
75% EtOH (in DEPC water)
RNase inhibitor
0.8M Sodium citrate/1.2M NaClO
DEPC water
50ml sterile plastic screw cap centrifuge tubes
Solutions or reagents:
TRIzol reagent
Phenol in saturated buffer(38%) : 380ml
Guanidine thiocyanate(0.8M) : 118.16g
Ammonium thiocyanate(0.4M) : 76.12g
Sodium acetate ph:5.0, (3M) : 33.4ml - stock (0.1M)
Glycerol(15%) : 50ml
 Procedure for RNA isolation:

*Grind 1g of chicken in liquid nitrogen

*Transfer powdered tissue to centrifuge tube containing trizol reagent
*Incubate sample at room temp for 5 min
*Homogenize tissue with homogenizer for 15 second
*Centrifuge at 1200RPM for 10 min
*Transfer the supernatant into new centrifuge tube and discord pellet
*Add chloroform and shake it for 15 sec
*Let the tube placed at room temp for 2-3 min
*centrifuge at 10,000RPM for 15 min
*Precipitate RNA by adding isopropanol and sodium citrate half volume of aqueos phase
*Cover the tube and mix it gently. Keep it at room temp for 10min
*Centrifuge tubes at 10,000RPM for 10min(discord supernant)
*wash the pellet with 20ml of 75% ethanol
*Centrifuge at 10,000RPM for 10min.Discard supernant and dry pellet.
*Add DEPC water to pellet resuspend by pipetting up and down
*Add R Nase inhibitor
*Transfer the sample into centrifuge tube at room temp.
*Centrifuge the sample at high speed for 5 min at room temp.
*Transfer RNA solution and store at 70% C
 Procedure for Gel electrophoresis

1. Use a 250 mL flask to prepare the gel solution. To the flask add 0.24 g of agarose, 0.6 mL concentrated (50x) buffer, and
29.4 mL distilled water. You will have a total volume of 30 mL. Swirl the mixture to disperse any clumps of agarose powder.

2. Us a marking pen to indicate the level of the solution volume on the outside of the flask.

3. Cover the flask with plastic wrap to minimize evaporation. Place the flask in a microwave oven and heat on high for 1
minute. Swirl the mixture and continue heating on high in bursts of about 25 seconds until all the agarose has dissolved.

4. Coll the agarose solution to 55 oC with careful swirling to promote even dissipation of heat. Make sure that the level of
the liquid is the same as before heating. If not, add distilled water to bring the volume up to the original volume.

5. Pull up the dams on the ends of a 7 x 7 gel bed. Place the bed on a level surface, add a six tooth comb and pour the
solution into the bed.
6. Let the gel completely solidify. It will be firm and cool to the touch. This will take approximately 20 minutes.

Preparing the Gel of Electrophoresis:

1. Drop the gates and carefully remove the comb by slowly pulling it straight up. You do not want to tear the sample
wells.

2. Place the gel bed into the electrophoresis chamber