Northern tRNA charging assay

tRNA Northern Protocol (Natasha Pavlova, Thompson Lab)

1. Day 0: plate two sets of cells in 6 cm dishes (0.75M/dish for MEFs) – one for RNA, one for cell counting. For control sample, include one extra plate to be used for deacylation control.
2. Day 1: treat as planned, harvest RNA into 1 mL Trizol.
3. Count the companion set of plates, record biomass.
4. Proceed with Trizol-chloroform extraction and EtOH precipitation o/n.
5. Day 2: resuspend in tRNA reprecipitation buffer (0.3M acetate buffer, pH = 4.5 + 10 mM EDTA). and reprecipitate in EtOH.
6. For deacylation control, resuspend in 150 uL Tris pH=9.0; incubate at 37C for 50 min, quench with equal volume of tRNA quench buffer (50 mM Na Acetate buffer (pH = 4.5) + 100 mM NaCl) and EtOH-precipitate.
7. Day 3: pour gels using the BioRad system (Helin lab). Wash the equipment with ddH₂O, spray with 95% EtOH and wipe, watch out for lint. For communal equipment, wash with dish soap, rinse really well with dH₂O, then ddH₂O, wipe dry, then 95% EtOH, wipe dry.
8. Assemble the casting rig using 1.0 mm glass plates and test for leaks with 100% EtOH.
9. Prepare the casting solution. In the fume hood, polymerize with 124 uL 10% APS (ammonium persulfate) and 34 uL TEMED, pour into gel rig. Insert comb, let polymerize for 30 min.
10. Spin down tRNA samples for 30 min; wash once with 80% EtOH and resuspend in 20 uL tRNA resuspension buffer (10 mM acetate buffer pH = 4.5 + 1 mM EDTA).
11. Prepare samples – normalize RNA volume by biomass, bring up with tRNA resuspension buffer, mix 1:1 with 2x RNA loading buffer. Include ssRNA ladder sample: 2 uL of NEB ssRNA ladder + 18 uL tRNA resuspension buffer + 20 uL 2x RNA loading buffer.
12. Pre-chill the running buffer beforehand. Pre-rinse the wells with the running buffer immediately before loading. Load the samples (~20 uL per lane). Run in cold room with a stir bar, 84V for ~5 hours. 
13. Disassemble and pour out buffer at the 4-hr mark, mix, then reassemble.
14. Disassemble the gel, stain in 1:10,000 SybrGOLD in 0.5x TBE in a 15 cm TC dish for 40 min.
15. Rinse the gel in 0.5x TBE.
16. Set up transfer in the BioRad transfer assembly, run for 2 hrs in 4C, 40V.
17. UV crosslink the membrane in the Stratalinker at 1200 μJoules; place the membrane on top of one of the still-wet filter papers used in transfer. Do not inhale when opening Stratalinker (ozone is formed).
18. Pre-warm the shaker oven, then dry the membranes in it at 50°C for 20 minutes.
19. Block the membrane at 42°C in the shaker oven in Church buffer (pre-warmed) with 10 ug/mL salmon sperm DNA 4 hours to overnight.
20. Incubate the membrane at 42°C in the shaker oven in Church buffer (pre-warmed) with 10 ug/mL salmon sperm DNA and 100 nM biotin-labeled oligo probe overnight, parafilm to prevent evaporation, also put a beaker with water inside the shaker oven, secure it with tape.
21. Day 5: wash the membrane 3 times with tRNA Northern wash buffer, incubate with Church buffer + salmon sperm DNA for 30 min at RT.
22. Switch to fresh Church buffer + salmon sperm DNA + streptavidin-HRP (1:7,500 dilution) for 30 min at room temperature.
23. Wash the membrane 3 times with tRNA Northern wash buffer.
24. Apply 4 mL ECL per membrane, develop.
25. Strip the membrane by dousing it with boiling 0.1% SDS in a glass IHC vessel, shake 15 min at RT.
26. Reblock in Church buffer (pre-warmed) with 10 ug/mL salmon sperm DNA for 4 hrs at 42C, shaking, then repeat steps 20-22 as necessary
27. If needed, store the membrane at 4°C in wash buffer or dry and store at RT.

Buffer Recipes

DEPC-treated water

Add 1 mL DEPC to 1L ddH₂O, incubate 3 hrs in the 37°C incubator with occasional mixing by inversion, then autoclave for 40 min.

Running Buffer (1L, keep cold, do not reuse)

0.3M Na Acetate pH=5.0 in DEPC-treated water (300 mL of 1M Na Acetate pH=5.0 + 700 uL DEPC water).

2x RNA loading buffer

4.2 G urea

3.0 mL 1M Na Acetate pH=5.0

Heat to 70°C in water bath to dissolve urea

Bring up to 10 mL with RNAse-free water

Check pH (~5.0) with a strip, adjust with glacial acetic acid if necessary (didn't need to)

Add 500 uL glycerol (5% final)

Add 500 uL 1% xylene cyanol and 1% bromophenol blue.

Casting Solution (6.5% gel)

4.2 G urea

3.0 mL 1M Na Acetate pH=5.0

Heat to 70C in water bath to dissolve urea

Add 1.7 mL 40% polyacrylamide (19:1)

Check pH (~5.0) with a strip, adjust with glacial acetic acid if necessary (used ~40 uL)

10% Ammonium Persulfate

Dissolve 1 G ammonium persulfate in 10 mL water and keep at 4°C (good for 2 weeks).

Transfer Buffer (1L, keep cold, do not reuse)

0.41G sodium acetate

10 mL 1M Tris-acetate pH=7.8

1 mL 0.5M EDTA, pH=8.0

1M Na Acetate buffer, pH = 5.0 (1 L)

82G sodium acetate in 800 mL DEPC water

pH with glacial acetic acid to pH = 5.0 (50+ mL of acid needed)

top with DEPC water to 1 L

filter

1M Tris-Acetate buffer, pH = 7.8 (0.5 L)

60.5G of Tris in 400 mL DEPC water

pH with glacial acetic acid to pH=7.8

top with DEPC water to 500 mL

filter

5X TBE (50 mL)

2.7 G Tris

1.375 G Boric acid

1 mL 0.5M EDTA, pH=8.0

DEPC water to 50 mL

filter

Modified Church Buffer for Hybridization (150 ml)

7% SDS (10.5 G, wear N95 if using powder)

200 mM Na₂HPO₄ pH=7.0

10 ug/mL salmon sperm DNA (add right before use, 1:1000 from stock)

Na₂HPO₄ pH=7.0

0.2M Na₂HPO₄: dissolve 2.84 G Na₂HPO₄ in 100 mL water

0.2M NaH₂PO₄: dissolve 2.76 G Na₂HPO₄* H₂O in 100 mL water

to 100 mL 0.2M Na₂HPO₄, add ~58 mL of 0.2M NaH₂PO₄ to pH = 7.0

filter

tRNA Northern Wash Buffer (500 mL)

25 mL 20x SSC

5 mL 10% SDS

470 mL DEPC water

Category