Northern Blot for tRNA

 

 

RNA extraction

 

All steps should be carried out in the cold.

 

1. Grow MEF cells in five 100-mm dishes.

 

2. Wash cells 2x with cold PBS and harvest by microcentrifuging (1,200 rpm, 4°C, 5 min).

 

3. Resuspend the cell pellet with 0.3 ml of 0.3 M sodium acetate (pH 5.0), and 10 mM EDTA.

 

4. Add same volume of phenol equilibrated with the same buffer.

 

5. Vortex 3x for periods of 30, 60, and 60 seconds with 60-s intervals between each step.

 

6. Microcentrifuge (14,000 rpm, 4°C, 5 min) and transfer the upper layer (aquous phase) to new tubes containing 0.3 ml of phenol equilibrated with the same buffer.

 

7. Vortex for 60-s, and microcentrifuge (14,000 rpm, 4°C, 5 min).

 

8. Transfer the upper layer to new tubes, and add 2.5 volumes of ethanol, and leave on ice for 1-2h.

 

9. Microcentrifuge (14,000 rpm, 4°C, 15 min), and dissolve the pellet with 60 ul of 0.3 M sodium acetate (pH 5.0).

 

10. Precipitate nucleic acids with 2.5 volume of ethanol, leave on ice for 2-3 h, and microcentrifuged (14,000 rpm, 4°C, 5 min).

 

11. Dissolve the pellet with 20 ul of 10 mM sodium acetate (pH 5.0), and 1 mM EDTA.

 

12. Use an aliquot (1.5 ul) to measure RNA amount at 260 nm and an aliquot (1 ul) to estimate the quality of the tRNA preparation by electrophoresis on 1.2% agarose gel containing ethidium bomide (1 ug/ml).

13. Take aliquots and store them at –80°C.

 

Deaminoacylation

 

1. Mix an aliquot of nucleic acids (30-50 ul) with 250 ul of 0.2 M Tris-HCl, pH 8.0, and extracted with 300 ul of phenol equilibrated with the same buffer.

 

2. Vortex for 60-s, and microcentrifuge (14,000 rpm, 4°C, 5 min).

 

3. Transfer the upper phase to a new tube and precipitate nucleic acids with sodium acetate, pH 8.0 (half volume of upper phase) and ethanol (2.5 volume of total), leave on ice for 2-3 h, and microcentrifuged (14,000 rpm, 4°C, 15 min).

 

4. Dissolve the pellet with 100 ul of 0.1 M Tris-HCl, pH 9.0, and incubated at 37°C for 20 min to deacylate the tRNAs.

 

5. Precipitate the nucleic acids with 2.5 volume of ethanol, leave on ice for 2-3 h, and microcentrifuged (14,000 rpm, 4°C, 15 min).

 

6. Dissolve the pellet with 20 ul of 10 mM sodium acetate, 1 mM EDTA (pH 5.0) prior to dilution with acid gel loading buffer.

 

 

Electrophoresis

 

1. Mix 10 ul of RNA and 15 ul of sample buffer (0.1 M sodium acetate, pH 5.0, 8 M urea, 0.05% bromphenol blue, and 0.05% xylene cyanol)

 

2. Prepare a 0.4-mm-thick 6.5% polyacrylamide gel (19:1 acrylamide:bisacrylamide) containing 8M urea in 0.1M sodium acetate.

 

2. Perform electrophoresis at 500 V (about 12 V/cm) in a cold room until the bromphenol blue dye reaches the bottom of the gel (20-24 h).

 

3. Excise the portion of the gel between the xylene cyanol and bromphenol blue dyes, which contained tRNAs of interest

 

4. Electrotransblot onto a Nytran membrane using an Electroblot apparatus at 20V for 90 min with 40 mM Tris-acetate, 2 mM EDTA, pH 8.1 as transfer buffer.

 

5. Rinse the membrane briefly with 4 x SET, and then baked at 70°C for 2-3 h.

SET = 0.15 M NaCl, 0.03 M Tris-HCl, 2 mM EDTA, pH 8.0

 

Hybridization

 

1. Prehybridize the membrane at 42*C for about 4 h in a solution (15 ml/17 x 13-cm membrane) consisting of 4 x SET, 250 ug/ml sheared and denatured salmon sperm DNA, 0.1% SDS, and 10 x Denhardt’s solution.

1 x Denhardt’s solution = 0.02% BSA, 0.02% polyvinylpyrrolidone 40,

and 0.02% Ficoll)

 

2. Hybridize the membrane at 42*C overnight in the same solution (10 mL) with 5’-32P-labeled oligonucleotide probes.

 

3. Wash the membrane 4 x for 30 min each at room temperature with 3 x SET and 0.1%

SDS.

 

4. Autoradiograph.