Acid urea polyacrylamide gel electrophoresis (AUPAGE) and Northern analysis

Abstract 

This AUPAGE and Northern analysis is a technique to quantify the charging level of transfer RNAs (tRNAs) in cells. The charging level of tRNAs in a cell can change in various cellular conditions such as nutrition starvation or oxidative stress, affecting translation efficiency of mRNA on the ribosome. As the charged aminoacyl moiety on tRNA is labile in neutral pHs, all steps of analysis of tRNA charging level should be maintained in an acidic condition. In this protocol, the tRNA pool extracted in an acidic condition will be run on an acid urea gel at low temperature, maintaining the cellular state of charging level, to separate the charged from the uncharged species. The tRNA of interest will be then detected by a specific probe in a Northern blot analysis, providing information on the ratio of the charged and uncharged species. This protocol is particularly useful for analysis of prolyl-charged tRNA species, as the prolyl moiety is the most labile among all aminoacyl groups. 

Materials and Reagents

1. General reagents: trichloroacetic acid (TCA), sodium chloride (NaCl), sodium acetate (NaOAc), Ethylenediaminetetraacetic acid (EDTA), ethanol, isopropanol, sodium citrate,and sodium dodecyl sulfate (SDS)
2. 10% TCA solution
3. Phenol-chloroform-isoamyl alcohol (PCI; 83:17:3.4), pH 5.0
4. RNA extraction buffer: 0.3 M NaOAc, pH 4.5 and 10 mM EDTA
5. RNA wash buffer: 70% ethanol and 30% 10 mM NaOAc, pH 4.5
6. Gel-loading buffer: 10 mM NaOAc, pH 4.5 and 1 mM EDTA
7. Urea (Research Product International, Cat # 57-13-6)
8. 40% polyacrylamide/Bis 19:1(Fisher, Cat # BP1406-1)
9. Ammonium persulfate (APS; Fisher, Cat # BP179-100)
10. Tetramethylethylenediamine (TEMED; Fisher, Cat # BP150-100)
11. Bromophenol blue (BPB; Thermo Fisher Sci, Cat # A18469.09)
12. Xylene cyanol (XC; Thermo Fisher Sci, Cat # 422691000)
13. Tris-Borate-EDTA (TBE) Buffer, pH 8.0 (10X) (Thermo Fisher Sci, Cat # B52)
14. Electrophoresis buffer: 0.1 M Sodium acetate, pH 5.0
15. Nitrocellulose membrane (Cytiva, Cat # RPN203B)
16. Northern hybridization buffer: 0.9 M NaCl, 90 mM Tris-HCl (pH 7.5), 6 mM EDTA, 0.3% SDS, and 1% dry milk
17. DNA probe specific to the target RNA, with a radioactive label such as ³²P, or a fluorescent label such as fluorescein amidites (FAM)
18. 2X SSC buffer: 0.3 M NaCl and 30 mM NaOAc
19. 2X acidic loading buffer: 9 M Urea, 0.1 M NaOAc (pH 5.0), 0.05% BPB, and 0.05% XC
20. Whatman membrane
21. Glass plates for intermediate size (14 x 17 x 0.05 cm) and for mini-size (8.6 x 6.7 x 0.1 cm) and gel running apparatus (Life Technologies, Gibco BRL Model V16)
22. 0.5 mm spacers and 18-well gel combs

Equipment and Software

1. Savant SpeedVac vacuum concentrator (Thermo Fisher Sci, Cat # DNA110-120)
2. UV crosslinker (Fisher, Cat # FB-UVXL-1000)
3. Trans-Blot Turbo Transfer system (Bio-Rad, Cat # 1704150)
4. Phosphorimager (GE, Amersham Typhoon model 9500)
5. ImageJ Software (NIH)

Procedure

A. Cell culture and RNA preparation.

Note: this is an example for 20 mL E. coli culture harvested at OD₆₀₀ = 0.4-0.5.

1. Pre-warm 20 mL 10% TCA at 37 °C prior to cell harvest.
2. Grow the cell to the desired OD₆₀₀.
3. At the time of harvest, pour 20 mL 10% TCA into the 20 mL cell culture, and swirl-mix.
4. Transfer the mix to ice and incubate for 10 min.
5. Spin down cells at 7,500 xg for 5 min at 4 °C.
6. Resuspend the pellet in 600 µL of the extraction buffer, while keeping samples chilled as much as possible.
7. Mix the cell resuspension with 600 µL of pre-chilled PCI.
8. Vortex the mixture for 5 cycles of 1 min vortex and 1 min rest on ice.
9. Spin at 13,500 xg for 10 min at 4 °C.
10. Take the top layer of the RNA-containing aqueous phase and mix with an equal volume (~600 µL) of cold isopropanol in a clean Eppendorf tube.
11. Incubate at −20 °C for 30 min.
12. Spin at 17,000 xg for 20 min at 4 °C.
13. Wash the pellet with 500 µL of the wash buffer, spinning at 17,000 xg for 5 min at 4 °C.
14. Dry the pellet by SpeedVac without heating. The samples can be stored at −80 °C, but it is highly recommended to proceed to the gel analysis immediately. Some aminoacyl tRNAs, such as Pro-tRNA^Pro, are highly labile and may be deacylated after freeze-thaw.
15. Dissolve the pellet in ~10 µL gel-loading buffer. Measure the concentration of RNA.

B. Gel preparation and setup.

Note: the intermediate-size gel was used specifically for analysis of Pro-tRNA^Pro. A mini-size gel can be used for other tRNAs. See the original publication Masuda et al., eLife (2021).

1. Weigh 10.5 g of urea in a 50 mL falcon tube. 

2. Add 4.06 mL of 40% polyacrylamide/bis 19:1 and 2.5 mL of 1 M NaOAc (pH 5.0) to the falcon tube.

3. Add autoclaved distilled water to bring the volume up to 25 mL. The gel solution will contain 7 M urea, 6.5% polyacrylamide/bis 19:1, and 0.1 M NaOAc pH 5.0.

4. Mix the solution to dissolve urea completely by shaking in an angular rotor for 20-30 min at room temperature. As this is an endothermic reaction, the solution gets cold. 

5. Set up the glass plates. Clean the intermediate size plates of 14 x 17 x 0.05 cm with dish soap and then with 70% ethanol. Dry the glass plates.

6. Clean two thin spacers of 0.5 mm and an 18-well comb.

7. Lay down the longer plate on the bottom once the plates are completely dried. Set up the spacers on the edge of the plates. It is helpful to wet the spacers to stick it properly on the plate. 

8. Place the shorter plate on the top of the longer plate and spacers. Make sure that the edges of the plates are properly aligned to prevent leakage.

9. Clamp the sides of the plates 5 cm above from the bottom. Tightly seal the bottom of the plates with a shipping tape. Two layers of taping can be used to prevent leakage.

10. Clamp the plates with 3 large paper clips on each side. 

11. Polymerize the gel solution. Make fresh 10% APS by dissolving 0.5 g of APS in 500 µL of autoclaved dH₂O. Do not use the old stock. To the 25 mL of gel solution from step B4, add 200 µL of 10% fresh APS and 15 µL of TEMED.

12. Mix by inverting the tube a few times and pour the gel solution into the gel cassette set up in step B10. Add the gel solution slowly by using a 1 mL pipette and avoid any bubbles being trapped inside the gel

13. Insert the clean comb slowly preventing any air bubbles. Keep the gel cassette in a horizontal position after inserting the comb. Put some weight on the top of the glass plate, especially near the comb to prevent the formation of a gel layer inside the wells.

14. Allow the gel to polymerize for >3 hours or overnight.

15. Set up the running apparatus. After the gel has polymerized, remove the clamps from both sides of the plates and the shipping tape from the bottom of the plate.

16. Before removing the comb, add some dH₂O in the upper part of the gel. Remove excess gel from the region using a clean blade and gently pull the comb out of the plate. Be very careful while removing the comb as it can tear the wells very easily.

17. Clamp the plate to the running apparatus.

18. Add electrophoresis buffer to the top and bottom chambers (~ 250 mL in each chamber) of the running apparatus. 

C. Running the AUPAGE.

1. Flush the wells thoroughly using a syringe for 5-10 times.

2. Mix 7 µg of total RNA (with the estimation that tRNA represents 10% of the total RNA) with an equal volume of 2X acidic loading buffer. Do not heat the samples.

3. Load the samples (6 µL maximum) slowly. Make sure to flush the well immediately before loading each sample.

4. Connect the running apparatus to the power supply and run the gel at 250 V at 4 °C for 3 h 45 min until the BPB dye front is near the bottom of the gel, but before it runs off the gel. 

D. Northern blot analysis.

1. After electrophoresis, remove the gel sandwich from the apparatus.
2. Cut the gel between BPB and XC, where tRNAs have migrated.
3. Soak the gel piece in 1X TBE buffer for 5 min while it is still on one of the plates.
4. Soak the nitrocellulose membrane in 1X TBE buffer in a separate container.
5. While the gel and membrane are in soaking, assemble the transfer apparatus: wet 8 pieces of Whatman filter paper according to the size of gel to be transferred to 1X TBE buffer.
6. Place 4 pieces on the semi-dry transfer apparatus; roll a plastic roller over the wetted filter paper to remove air bubbles.

7. Place the soaked nitrocellulose membrane on the top of the gel and transfer the gel from the plate to the pre-soaked nitrocellulose membrane gently. Use new gloves while performing this transfer. Caution: The gel is very thin (~0.5 mm), which is likely to tear apart during this transfer step.

8. Place the nitrocellulose membrane with the gel on the Whatman filter paper in the transfer apparatus.

9. Place the remaining 4 wet Whatman filter paper on the top of the gel.

10. Roll over the sandwich to remove the remaining air bubbles. Use a piece of large Kim-wipe paper to remove excess transfer buffer.

11. Close the transfer cassette and start the semi-dry transfer at 25 V constant, 1.0 mA for 20 min.

12. Disassemble the transfer apparatus. Remove the electroblotted membrane and briefly air-dry it.

13. Crosslink the RNA on the membrane using an ‘optimal crosslink’ in a UV crosslinker. It usually takes less than 1 min.

14. Pre-incubate the membrane in a hybridization buffer at 37 °C for 1 h.

15. Add 10⁶ cpm of the ³²P-labeled DNA probe at the 5'-end that is specific for the targeted tRNA. To avoid using radioactive material, the probe can be prepared with fluorescent labels, such as FAM.

16. Incubate the probe with the membrane in the hybridization buffer for 12 h while shaking at 37 °C.

17. Wash the probes off by 2X SSC buffer at 37 °C two times, each time with mild shaking for 10 min.

18. Dry the membrane and expose it to an imaging plate overnight.

19. Visualize the signal by a phosphorimager instrument and quantify the band using ImageJ software. Representative gel images can be found in the references below.

References

1. This protocol was developed based on the method described in:

Parker et al., Cell Syst. (2020) DOI: 10.1016/j.cels.2020.07.005

2. This protocol was developed in:

Masuda et al., eLife. (2021) DOI: 10.7554/eLife.70619

3. Another representative gel image is found also in:

Masuda et al., Cell Rep. (2022) DOI: 10.1016/j.celrep.2022.111539