RNA Extraction Procedure Based on the Boom Method 15 see Note

1. Add 40 ^L of silica solution to 900 ^L of L6 buffer in a 1.5-mL microcentrifuge tube (see Notes 10 and 11).

3. Vortex the mixture for 5 s, keep it at room temperature for 10 min, and mix again for 5 s.

4. Centrifuge at 12,000g for 15 s and discard the supernatant (see Notes 10 and 13).

5. Wash the silica pellet twice with 900 ^L of L2 buffer, twice with 900 ^L of 70% ethanol, and once with 900 ^L of acetone. Centrifuge for 15 s at 12,000g after each wash and discard the supernatants (see Notes 10, 12, and 13).

6. Place the tube, with the lid open, at 56°C in a dry heating block for 10 min.

7. Add 50 ^L of TE buffer, vortex, and incubate for 10 min at 56°C with the lid closed (see Notes 10 and 12).

8. Vortex and centrifuge the tube at 12,000g for 2 min.

9. Recover the nucleic acid-containing supernatant in a new tube (see Notes 13 and 14) and store it at 4°C (see Note 15).

3.3. Two-Step RT-PCR for the Amplification of a Fragment of the 5'NCR and Confirmation by Southern Blot for the Generic HAV Detection (see Note 16)

1. Add 10 ^L of the extracted RNA to a 0.2-mL PCR tube.

2. Incubate the tube at 99°C for 5 min to denature the RNA.

3. Chill the tube on ice.

4. Prepare the RT mix (volume for one sample): 5.000 ^L 5X RT buffer; 0.125 ^L 100 ^M primer HAV 240; 2.500 ^L 2 mM dNTPs; 0.040 ^L M-MLV reverse trascriptase (200 U/ ^L); 7.300 ^L Distilled H2O; for a total of 15.000 ^L.

5. Add the 15 ^L of the RT mix to each tube containing denatured RNA.

1. Add 29.5 ^L of distilled H2O to a 0.2-mL PCR tube.

2. Prepare the PCR mix (volume for one sample): 5.00 ^L 10X Expand High Fidelity-MgCl2 buffer; 0.25 ^L 100 ^M primer HAV 240; 0.25 ^L 100 ^M primer HAV 68; 5.00 ^L 2 mM dNTPs; 0.14 ^L Expand High Fidelity PCR enzyme (3.5 U/^L); for a total of 10.50 ^L.

3. Add the 10.5 ^L of the PCR mix to each of the water-containing tubes and 10 ^L of the RT product.

4. Transfer the samples to the thermocycler and run the following program: 4 min at 94°C; 40 cycles of 1 min at 94°C, 1 min at 55°C, 1 min 30 s at 72°C; and 10 min at 72°C.

3.3.3. Electrophoresis

1. Mix 10 ^L of the PCR product with 2 ^L of 6X loading buffer and dispense them in a well of a 1.5% TBE-agarose gel. In a parallel well dispense 5 ^L of the working solution of the DNA molecular weight marker IX (Roche).

2. Connect the electric power at 70 V and run the samples for approx 2 h in TBE buffer.

3. Stain the DNA-containing agarose gel with a 1:10,000 dilution of GelStar in TBE buffer (see Note 17).

4. Analyze the DNA amplimers by observation of the stained bands in the gel analyzer using the UV light tray (see Note 18).

3.3.4. Southern Blot Hybridization

1. Transfer the gel to a glass Petri dish and submerge it in several volumes of 150 mM NaOH for 15 min to denature the double-stranded DNA. Keep the glass dish containing the gel on ice with constant shaking.

2. In the mean time, cut the nylon membrane (see Note 19) and rinse it in 0.5X TBE for 15 min. Wet fiber pads and filter papers with 0.5X TBE.

3. Wash and neutralize the gel by immersion in several volumes of 0.5X TBE. Keep the glass dish containing the gel on ice and shake constantly for 10 min.

4. Prepare the transfer cell as depicted in Fig 1. The gel should be in direct contact with the nylon membrane by the open end side of the well (see Note 20).

5. Close the cassette, and place it in the module.

6. Place 0.5X TBE buffer in the module.

7. Add the frozen Bio-Ice cooling unit.

8. Add a standard magnetic stir bar to maintain the buffer temperature and ion distribution in the tank.

9. Put on the lid, plug the cables into the power supply, and run for 1 h at 60 V.

10. Disassemble the blotting sandwich and remove the membrane.

11. UV-crosslink the wet membrane at 366 nm for 3 min.

12. Bake in a vacuum oven at 120°C for 30 min (see Note 21).

13. Place the membrane in a bag containing 20 mL of prehybridization solution per 100 cm2 of membrane surface area.

14. Seal the bag and prehybridize overnight at 40°C.

15. Discard the prehybridization solution and add the hybridization solution containing the DIG-labeled probe at a 5 pmol/mL concentration and at the same volume/surface ratio.

17. Place the membrane in a glass dish and wash it briefly in several volumes of 2X SSC at room temperature with constant shaking.

18. Wash the membrane for 15 min in several volumes of 2X SSC-0.1% SDS at room temperature with constant shaking.

19. Incubate the membrane for 1 min in several volumes of buffer I at room temperature with constant shaking.

20. Block the membrane by gentle agitation in several volumes of blocking solution for 30 min at room temperature (see Note 22).

21. Dilute the anti-dig-AP 1:10,000 in blocking solution and mix.

22. Discard the blocking solution and incubate the membrane for 30 min in the antibody solution. Use a standard Petri dish for the incubation and the same volume/surface ratio as with the hybridization solution (see Note 23). Gentle agitation of the dish is required to ensure a constant and complete contact of the antigen-bound membrane and the antibody-containing solution.

23. Discard the antibody solution.

24. Wash the membrane twice (15 min per wash) in several volumes of washing buffer.

25. Equilibrate the membrane in several volumes of phosphatase buffer for two minutes.

26. Prepare the CSPD solution at a 1:100 in phosphatase buffer.

27. Discard the phosphatase buffer, avoiding, however, drying of the filter.

28. Transfer the membrane into a plastic bag with CSPD solution at the same volume/surface ratio as before. Remove any bubble. Protect the bag from the light.

29. Incubate the bag at room temperature for 5 min with constant shaking.

Fig. 1. Southern blot transfer cell.

30. Transfer the membrane to a new plastic bag (see Note 24).

32. The chemilumininescent signal detection is performed by exposing the photographic film to the membrane-containing bag in the autoradiografic cassette (see Note 25).

34. Photographic development: transfer the film into several volumes of developer solution for 3 min. Rinse the film with water, transfer the film to several volumes of fixative solution for 3 min, and finally rinse the film again in water and let dry.

3.4. HAV Genotyping by Two-Step RT-PCR Amplification and Sequencing of a Fragment of the VP1X2A Junction Region

1. Add 10 ^L of the extracted RNA to a 0.2-mL PCR tube.

2. Incubate the tubes at 99°C for 5 min to denature the RNA.

3. Chill the tubes on ice.

4. Prepare the RT mix (volume for one sample): 5.000 ^L 5X RT buffer; 0.125 ^L 100 ^M primer VP1-3285; 2.500 ^L 2 mMdNTPs; 0.040 ^L M-MLV reverse trascriptase (200 U/ ^L); distilled H2O 7.300 ^L; for a total of 15.000 ^L

5. Add 15 ^L of the RT-mix to each tube containing the denatured RNA.

2. Prepare the PCR mix (see Note 27) (volume for one sample): 5.00 ^L 10X PCR-MgCl2 buffer; 0.25 ^L 100 ^M primer VP1-2949; 0.25 ^L 100 ^M primer VP1-3285; 5.00 ^L 2 mM dNTPs; 0.14 ^L Expand High Fidelity PCR enzyme (3.5 U/^L); for a total of 10.50 ^L.

3. Add 10.5 ^L of the PCR mix to each water-containing tube and 10 ^L of RT product.

4. Transfer the samples to the thermocycler and run the following program: 4 min at 94°C; 40 cycles of 1 min at 94°C, 1 min at 55°C, 1 min 30 s at 72°C; and 10 min at 72°C.

3.4.3. Electrophoresis

1. Mix 10 ^L of the PCR product with 2 ^L of 6X loading buffer and put them over a well of a 0.8% TBE-agarose gel. In a parallel well, dispense 5 ^L of the working solution of the DNA molecular weight marker IX (Roche).

2. Connect the electric power up to 70 V and run the samples for approx 2 h in TBE buffer.

3. Stain the DNA-containing agarose gel with a 1/10,000 dilution of GelStar in TBE buffer (see Note 17).

4. Analyze the DNA amplimers by observation of the stained bands in the ImageMaster, using the UV light tray (see Note 18).

3.4.4. DNA Purification

3.4.4.1. Purification of an Amplimer From Single Band-Containing Solutions

1. Mix the remaining 40 ^L of the PCR product with 200 ^L of the Binding Buffer provided with the kit.

2. Transfer these 200 ^L to the upper reservoir of a filter-tube inserted into a collection tube, both provided with the kit.

3. Centrifuge the combined tubes for 1 min at room temperature at 16,000g and discard the flowthrough solution.

4. Add 500 ^L of wash buffer to the upper reservoir (1:5 dilution of the 5X wash buffer provided with the kit in 96° ethanol).

5. Centrifuge for 1 min at room temperature at 16,000g and discard the flowthrough solution.

7. Centrifuge for 1 min at room temperature at 16,000g and discard the flowthrough solution.

8. Centrifuge for 5 s at room temperature at 16,000g.

9. Insert the filter-tube into a standard microcentrifuge tube.

10. Add 60 ^L of the elution buffer (provided with the kit) to the filter-tube, and incubate for 3 min at room temperature.

11. Centrifuge the combined tubes for 1 min at room temperature at 16,000g and keep the DNA-containing flowthrough solution.

12. Estimate the quantity of DNA by comparison of its fluorescence intensity with that of the 360-bp band of the DNA Molecular Weight Marker IX (Roche; 1.425 ng/^L) run in parallel in an agarose gel and stained with GelStar.

3.4.4.2. Purification of an Amplimer From Mixed Band-Containing Solutions

1. Run an electrophoresis of the remaining 40 ^L of PCR product, as previously described in Subheading 3.4.3. but using a 0.8% TAE-agarose gel instead of TBE-agarose. Prepare the agarose gel with a preparative comb.

2. Cut the desired band from the agarose gel with an ethanol-cleaned scalpel (see Note 28).

3. Weigh a 1.5-mL microcentrifuge tube, add the excised agarose fragment to the tube, and weigh it again to deduce the fragment weight.

4. Add binding buffer at a ratio of 300 ^L/100 mg of agarose and mix vigorously.

5. Incubate at 56°C for a minimum of 10 min to dissolve the agarose. Mix vigorously every 2 min.

6. Add isopropanol at a ratio of 150 ^L/100 mg of agarose and mix vigorously.

7. Transfer this solution (up to 700 ^L) to the upper reservoir of a filter-tube inserted into a collection tube.

8. Centrifuge the combined tubes for 1 min at room temperature at 16,000g and discard the flowthrough solution. Repeat this step with any further volume exceeding 700 ^L.

9. Proceed as in the purification of an amplimer from single band-containig solutions from (Subheading 3.4.4.1.) step 4.

3.4.5. DNA Sequencing

1. Prepare the sequencing mix: 4.00 ^L Ready Reaction Mix (see Note 29); 0.64 ^L 5 ^M primer VP1-2949 (see Note 30); x.xx ^L 3-10 ng of purified DNA (see Note 31); x.xx ^L distilled water (see Note 31) for a total of 10.00 ^L.

2. Transfer the samples to the thermocycler and run the following program: 25 cycles of 10 s at 96°C; 5 s at 50°C; 4 min at 60°C.

3. Keep the reaction product at 4°C until further processing (see Note 32).

4. Prepare the precipitation mix: 10.00 ^L sequencing product; 64.00 ^L 95° ethanol; 26.00 ^L distilled water; for a total 100.00 ^L.

5. Mix vigorously by vortexing.

6. Allow to stand for 15 min at room temperature.

7. Centrifuge for 30 min at 16,000g and discard the supernatant.

9. Centrifuge for 5 min at 16,000g and carefully discard all the supernatant.

10. Keep the tube open at room temperature until the pellet is completely dried.

11. Electrophorese the sequencing product on the ABI Prism Analyzer for 4.5 h according to the manufacturer's instructions.

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