BZLF-1, BamH1 Z fragment, left reading frame; for other abbreviations, see Table 1 footnote.

BZLF-1, BamH1 Z fragment, left reading frame; for other abbreviations, see Table 1 footnote.

2.3.4. Visualization and Detection of PCR Products by Southern Blotting

1. 50X TAE: 968 g Tris, 228.4 mL glacial acetic acid, 148.8 g EDTA, up to 4 L with water.

2. 6X Sample buffer: 0.25% bromophenol blue, 0.25% Zylene cyanol, 30% glycerol.

3. One-phor-all buffer (Pharmacia).

4. 100-bp Ladder (Invitrogen).

5. Alkalization solution: 1 M NaCl (232 g/4L), 0.5 M NaOH (80 g/L) in water.

6. Neutralization solution: 1 M NaCl (232 g/4L), 1 M Tris-HCl (484 g/L), pH to 7.4, up to 4 L with water.

7. Supercharged Nytran (Schleicher and Schuell).

8. QIAquick Gel Extraction Kit (Qiagen).

9. Nuseive GTG agarose (BMA Bioproducts).

10. Seakem LE agarose (BMA Bioproducts).

13. Random Primed labeling kit (Roche).

14. Hybridization oven.

15. 100X Denhardt's solution: 10 g Ficoll 400, 10 g polyvinyl prolidone, 10 g BSA up to 400 mL with water.

16. 20X SSC: 701 g NaCl, 352.8 g sodium citrate up to 4 L with water.

18. Wash A: 150 mL 20X SSC, 6.25 mL 20% sodium dedecyl sulfate (SDS) up to 500 mL with water.

19. Trichloracetic acid at 5 and 10%.

21. Whatman filters (934-AH).

22. Kodak X-OMAT AR film.

23. Prehybridization solution: 6X SSC, 1X Denhardt's solution, 1% SDS.

24. Hybridization solution: 6X SSC, 50% formamide, 1% SDS, 2% dextran sulfate.

25. Semilog paper.

3. Methods

3.1. Sample Preparation

3.1.1. Cell Line Controls

Several control experiments demonstrating the ability to detect expression of each gene from a single cell should be performed before proceeding to the analysis of clinical samples. Cell line controls should also be included in each experiment thereafter. Prepare separate controls for each of the necessary cell lines: IB4, AKATA, and B958 (see Note 1).

1. Split cells the day before use.

2. Count and resuspend cells to 1 x 103 cells/mL in PBS/BSA.

3. Prepare 10-fold serial dilutions to give concentrations of 1 x 102 and 1 x 101 cell/mL.

4. Aliquot appropriate volumes of these dilutions into microcentrifuge tubes to give six to eight tubes each of 1, 5, 10, and 100 cells per tube (see Notes 2 and 3).

5. Count and resuspend EBV carrier cells to 5 x 106 cells/mL in PBS/BSA. Add 1 mL to each of the tubes prepared in step 4 (see Note 4).

6. Proceed to Subheading 3.3.

3.1.2. Purification of Mononuclear Cells Via Ficoll (see Note 5)

1. Turn centrifuge to 25°C, and turn off the brake; leave it off for entire purification.

2. Obtain heparinized blood through routine venipuncture (see Note 6).

3. Dilute blood 1:1 in 1X PBS.

4. Slowly layer 30 mL of diluted blood onto 20 mL of room-temperature Ficoll in a 50-mL conical tube. Be careful to maintain an interface between the two layers.

5. Place tubes in centrifuge, and spin for 30 min at 400g, 25°C.

6. Aspirate off plasma layer (top layer; yellow in color); be careful not to disturb the buffy coat layer (white interface between the plasma and Ficoll layers).

7. Carefully remove buffy coat cells with a sterile transfer pipet to a fresh 50-mL conical tube. Buffy coat cells from two tubes may be combined to a total volume of 50 mL in PBS/BSA.

8. Invert tubes to mix, and centrifuge at 4°C for 15 min at 350 g.

9. Aspirate supernatant.

10. Resuspend all pellets together to a total volume of 50 mL in PBS/BSA.

12. Centrifuge cells for 10 min at 4°C and 1300g.

13. Resuspend cells to 5 x 106 cells/mL in PBS/BSA.

14. From the total number of cells (see Note 8) available, determine a series of dilutions to be used for RT-PCR analysis (see Note 9) and a separate set of dilutions to be used for DNA PCR (see Note 10).

15. Aliquot the RT-PCR dilutions into microcentrifuge tubes, and add EBV- filler cells to each tube to give a total of 5 x 106 cells per tube (see Note 4). Proceed to Subheading 3.3.

16. Aliquot the DNA-PCR dilutions into a 96-well plate (see Note 11). Proceed to Subheading 3.2.

3.2. Quantitation of EBV-Infected Cells: DNA-PCR

1. Centrifuge microtiter plate for 15 min at 4°C, 400g.

2. Aspirate supernatant from each well, being careful to not dislodge the cell pellet

3. Prepare digestion buffer as follows: 100 |L 4.5% Tween-20, 100 |L 4.5% NP-40, 100 |L PCR buffer, 50 |L proteinase K (10 mg/ml; Invitrogen; see Note 12), and 650 |L of H2O.

4. Resuspend cell pellets in 10 |L of digestion buffer.

5. Incubate the microtiter plate at 55°C overnight (see Note 13).

6. Add 5 |L of water to each well; mix well. The samples are now ready for PCR.

Fig. 1. Limiting dilution DNA PCR for the determination of the frequency of EBV-infected cells. Example of a Southern blot for a limiting dilution DNA PCR for EBV in a population of B cells from the peripheral blood of an infectious mononucleosis (IM) patient. Note that the numbers of cells per well are low. This is because IM patients have very high frequencies of infected cells. If this were a frequency analysis for a healthy carrier, the cell dilutions would need to be much higher (103-106 per well).

7. Preheat the PCR machine to 95°C.

8. Count the number of sample wells, add 10 to this number, and call it x (see Note 14). Carefully label 0.2-mL thin-walled PCR tubes.

9. Prepare PCR master mix for x tubes as follows: 1x ||L 20 pM EM2 primer, 1x ||L 20 pM W1 primer, 1x |L 10 mM dNTP mix, 1x |L 25 mM MgCl2, 5x |L Taq Supplied Buffer, and 31x |L HPLC H2O.

10. Aliquot 40 |L of master mix into each PCR tube.

12. Heat samples for 10 min at 95°C.

13. Prepare Taq solution for X tubes as follows: 4.3x |L of H2O, 0.5x |L of Taq supplied buffer, and 0.2x ||L of Taq.

14. Add 5 |L of this mix to each tube while the tubes are still in the heat block.

15. Transfer samples directly from the heat block to a preheated PCR machine (see Note 15).

16. Perform amplification with the following program: 35 cycles at 95°C for 30 s, 65°C for 1 min and 1 cycle at 72°C for 5 min.

17. Visualize the PCR products on agarose gel according to the Southern blotting procedure detailed below in Subheading 3.4. Results are tabulated as described in Subheading 3.5. Figure 1 shows example results from this procedure.

3.3. Quantitation of EBV Gene-Expressing Cells: RT-PCR

3.3.1. RNA isolation

1. Turn on all heat blocks (55°C, 42°C, 68°C, 95°C).

2. Centrifuge samples, including controls, prepared in Subheading 3.1. at 300g for 5 min.

3. Pour off supernatant. Dab on a paper towel to remove as much liquid as possible.

4. Resuspend each sample in 1 mL of Trizol reagent (see Notes 16 and 17).

5. Let stand at room temperature for 5 min.

6. Add 200 ||L of chloroform, and shake vigorously for 15 s.

7. Let stand for 2 min at room temperature; you should see a clear layer on top and a pink layer on bottom. If the layers do not separate, shake again and let stand for 2 min.

8. Centrifuge at 12,000g for 15 min.

9. During the spin of step 5, prepare a new set of tubes (one for each sample), and add 500 |L of room-temperature isopropanol to each tube.

10. When the spin of step 5 is complete, carefully remove the clear aqueous layer from the top of each sample, and add this to the isopropanol (see Note 18).

11. Vortex each sample thoroughly and let stand at room temperature for 10 min.

12. Centrifuge at 12,000g for 10 min at 4°C.

13. Carefully pour off the isopropanol (see Note 19).

14. Add 1 mL of ice-cold freshly prepared 70% EtOH to each tube; vortex briefly.

15. Centrifuge samples at 7500g for 5 min.

16. Pour off EtOH at 4°C (see Note 20). 14. Air-dry for 8 min (see Note 21).

16. Add 7 | L of DEPC or HPLC H2O to each tube; do not pipet up and down.

17. Heat for 10 min at 55°C, then vortex gently, and return to 55°C for a further 5 min.

18. Quick-spin to gather liquid to the bottom of the tube.

19. Proceed immediately to cDNA synthesis to prevent RNA degradation.

3.3.2. cDNA Synthesis

1. Add 5 |L of random primers (50 ng/|L) to the bottom of one 0.2-mL PCR tube for each sample.

2. Add 7 |L of RNA, and mix gently by pipeting.

5. Quick-spin tubes to bring the total volume to the bottom of the tube.

6. Prepare First Strand Master Mix as follows. For one reaction: 4 |L of First Strand Buffer (Invitrogen), 2 |L of DTT (Invitrogen), and 1 |L of 10 mM dNTPs (Invitrogen).

7. Add 7 |L of First Strand Master Mix to each sample; mix gently with pipet tip, but do not pipet up and down.

8. Incubate at room temperature for 10 min.

9. Add 1 |L of Superscript, and mix with pipet tip; do not pipet up and down.

10. Incubate for 10 min at room temperature.

12. Stop reaction by incubation for 15 min at 68°C.

13. Add 180 |L of HPLC H2O, and vortex thoroughly; cDNA is now ready for PCR (see Note 22).

2. Carefully label one set of PCR tubes for each target gene. Include two water controls for each gene.

Fig. 2. Limiting dilution RT-PCR. (A) Results of a control experiment using EBV+ cell lines. This experiment demonstrates that each RT-PCR assay can detect RNA from a single infected cell. (B) and (C) Results of limiting dilution analysis performed on cells from infectious mononucleosis (IM) patients. For patient 1, the experiment was performed exactly as described in this chapter. For patient 2, B cells were first isolated by negative selection using the Stem Sep System from Stem Cell Technologies. For both patients it is clear that only a small fraction of cells is expressing each gene, as there are 100s of infected cells in each lane, yet many samples were negative for gene expression. The exact percentage of cells expressing each gene is indicated below the blots. For patient 1 the frequency of infected cells was determined to be 1 infected cell per 6250 peripheral blood mononuclear cells (PBMCs; data not shown), and for patient to the frequency was determined to be 1 infected cell per 45 B cells. BZLF, BamH1 Z fragment, left reading frame; EBER, Epstein-Barr virus-encoded small RNA; EBNA, Epstein-Barr nuclear antigen; LMP, latent membrane protein.

3. Prepare a separate master mix for each target gene. Make enough mix for the number of RT samples plus two H2O controls. For one PCR reaction: 1 ||L 20 pM forward primer, 1 |L 20 pM reverse primer, 1 |L 10 mM dNTPs (Invitrogen), (A)* |L MgCl2, 5 |L 10X Taq Supplied PCR Buffer, and (B)* |L of H2O. *, The volumes of MgCl2 and H2O are different for each target gene. The values for each are given in Table 3.

4. Aliquot 25 | L of the appropriate master mix into each PCR tube.

6. Incubate tubes for 5 min at 95°C.

7. Prepare Taq master mix for 10 tubes: 43 |L of HPLC H2O, 5 |L of Taq Supplied Buffer, and 2 |L of Taq.

8. Add 5 |L of Taq mix to each tube while it is still in the heat block.

9. Transfer tubes directly to a preheated PCR machine (see Note 15).

10. All PCR programs are as follows: 1 cycle at 95°C for 5 min and 40 cycles at 95°C for 15 s, annealing temperature for 30 s, 72°C for 30 s, and 1 cycle at 72°C for 5 min. Annealing temperatures for each gene can be found in Table 3.

11. PCR products are then visualized via the Southern Blotting protocol detailed below in Subheading 3.4. An example of results for a clinical IM sample and controls are shown in Fig. 2.

3.4. Visualization and Detection of PCR Products by Southern Blotting

3.4.1. Electrophoresis and Transfer of DNA

1. Prepare a 2% Nuseive/1% Seakem agarose gel in 1X TAE containing ethidium bromide.

2. Mix 12 | L of PCR product with 2 | L of sample buffer and incubate at 65°C for 5 min; prepare a 100-bp ladder by mixing 50 |L of the ladder with 25 |L of sample buffer and 25 | L of One-Phor-All buffer; do not heat the bp ladder.

Number of A) Cell Line Controls

Infected Cells


i i i I i i i i i I i I Ii i i i I I I i i I I I i i I i i i i i i i i

Number of PBMCs

Number of Infected Cells

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