A fluorescent in situ hybridization (FISH) assay for detection of HBV DNA in cell culture models

POSTED ON: 19 Jun, 2020

Sample preparation

  1. Cell culture and HBV infection.

1.1 Tetracycline-inducible (tet-off) HepAD38 were seeded on collagen-coated four-well chamber slides and maintained in culture medium for 48 – 72 h.

1.2 HepG2-NTCP cells were cultured in collagen-coated four-well chamber slides, and at approximately 100% confluence, concentrated supernatants of HepAD38 cells were mixed with culture medium containing 4% PEG8000 and inoculated onto cells for 6 – 8 h. The cells were subsequently washed three times with PBS and maintained in replication medium for several days based on the aim of experiment.

  1. Fix the cells in 3.7% formaldehyde solution and dehydration.

2.1 In a fume hood, prepare 6 ml of fresh 3.7% formaldehyde solution by diluting 500 ul of a 37% stock formaldehyde with 5.5 ml of PBS and then mixing well.

Caution: Formaldehyde is a poison and irritant. Avoid contact with skin and mucous membranes.

2.2 Gently aspirate off culture medium and rinse cells three times, each time with 300 ul/well of PBS.

  • Critical: Before fixation, cell attachment may be weak. Aspiration and dispensing of contents from and into the chamber should be performed slowly and gently with a 1 mL pipette.

Aspirate from the corners and dispense against the chamber wall.

2.3 Aspirate off the final PBS wash and add 300 μL/well of freshly prepared 3.7% formaldehyde solution. Cover chamber slide with lid and incubate at RT for 10 minutes.

2.4 Aspirate off the formaldehyde solution and gently rinse the cells three times, each with 300 ul/well of PBS.

2.5 Aspirate off the PBS and replace it with 300 μL/well of 50% ethanol. Incubate for 5 minutes at RT.

2.6 Aspirate off the 50% ethanol and replace with 300 μL/well of 100% fresh ethanol. Seal the chamber slides with parafilm and store the dehydrated cells in 100% ethanol at –20 °C until useda.

If doing the experiment right away, aspirate off the 50% ethanol and replace with 300 μL/well of 70% ethanol at 4 °C for at least 1 hour and proceed on 3.1.B

NOTE:

  1. Dehydrated cells can be stored under these conditions for up to one month and must be rehydrated before being used in the in situ assay.

FISH assay procedure

  1. Cell rehydration.

3.1 A. Aspirate off the 100% ethanol (corresponding to 2.6 A) and replace with 300 μL/well of 70% ethanol. Incubate for 5 minutes at RT.

B.  Aspirate off the 70% ethanol (corresponding to 2.6 B) and replace with 300 μL/well of 50% ethanol. Incubate for 5 minutes at RT.

C. Aspirate off the 50% ethanol and replace with 300 μL/well of PBS. Incubate for 10 minutes at RT.

3.2 Aspirate off the PBS and add 300 μL/well of a 3.7% formaldehyde solution. Incubate for 10 minutes at RT.

3.3 Aspirate off the formaldehyde solution and gently rinse the cells three times, each with 300 μl/well of PBS for 5 minutes at RT.

  1. Protease digestiona.

4.1 Prepare 1.2 ml diluted working Protease Solution (1:3000) in PBS by adding 0.4 ul Protease QSb to 1199.6 μl PBS. Vortex briefly to mix.

4.2 Aspirate off the PBS and added 300 μL/well of working protease solution. Cover chamber slides with lid and incubate for 10 minutes at 37 °C in a water bath.

4.3 Aspirate off the working protease solution from the chamber and rinse the cells three times, each with 300 μl /well of PBS.

4.4 Aspirate off PBS and add 300 μL/well of a 3.7% formaldehyde solution. Incubate for 5 minutes at RT.

4.5 Aspirate off the formaldehyde solution and gently rinse the cells three times, each with 300 μL/well of PBS.

NOTES:

  1. The Protease digestion step partially degrades capsid and polymerase that wrap HBV DNA thus increasing target accessibility. However the exact concentration of protease should be empirically determined because excessive digestion can cause complete release of nucleic acids and even cell morphology damage and cell loss.
  2. Protease QS is provided in the kit. Thaw and place Protease QS on ice until use.

 

  1. Digest with RNase A/Ha.

5.1 Prepare 1.2 ml diluted working RNase A/H solution by adding 2.4 μl RNase A 1:500 and 2.4 μL RNase H 1:500 to 120  μL 10X RNaseH Reaction buffer and 1075.2 μl deionized water.

5.2 Aspirate off the PBS and added 300 μL/well of working RNase A/H solution. Incubate the slides in a cassette for 1 hour at 37 °C on the water bath.

5.3 Aspirate off the working RNase A/H solution and gently rinse the cells three times, each with 300 μL/well of PBS for 5 minutes at RT.

5.4 Aspirate off the PBS and added 300 μL/well of 3.7% formaldehyde solution. Incubate for 5 minutes at RT.

5.5 Aspirate off the formaldehyde solution and gently rinse the cells three times, each with 300 μL/well of PBS for 5 minutes at RT.

NOTES:

  1. a. RNase A is an endonuclease that specifically degrades single-stranded RNA.

RNase H is an endonuclease, it can decompose the RNA strand in the RNA/DNA hybrids.

Our previous results suggested that HBV RNA could exist in the form of RNA/DNA hybrid in core particles1,3.  The combined use of RNase A/H digestion can increase signal and improve assay specificity. First, probe set 3 targets the plus strand of HBV DNA but can also bind to HBV RNA. RNase digestion eliminate residual HBV RNA in the replication complex and capsid-free HBV mRNA. Second, RNaseH digestion eliminate the HBV RNA that binds to minus strand DNA hence increasing the hybridization efficiency of probe set 2.

 

   6. Hybridization with Probe.

6.1 Prepare 150 μl  working Probe Set Solution by diluting 1.5 μl of each Probe Set (1:100) in 148.5 μl  pre-warmed Probe Set Diluent QFa. Gently mix the solution and shortly centrifuge 10-20 seconds. 150 µl for every slide?

Caution: Probe Set Diluent QF contains formamide, a teratogen, irritant and possible carcinogen. Avoid contact with skin and mucous membranes.

6.2 Remove the slides from the chamber and gently decant the remaining PBS, then put the slide edge on a clean, dry paper towel for 1-2 seconds. Add 150 μl/slide of the working Probe Set Solution to the slide, subsequently cover with a 24×50 mm2 coverslip and seal edges with rubber cement.

  • Critical: The chamber on the slide may not be easily removed. Handle with care to avoid the slide broken.

6.3 Preheat the cassette at 75 °C in the dry incubator for 2 minutes.

6.4 Then incubate at 40 °C for 3 hours in the water bath or the Thermobrite hybridizer.

NOTE:

  1. Pre-warm Probe Set Diluent QF to 40 °C in a water bath for 10 minutes to dissolve possible precipitates.

 

7. Hybridize with Pre-Amplifier

7.1 Prepare 15 ml Wash Buffer by adding 45 μl Wash Comp1 and 75 μl Wash Comp2a to 14.88 ml deionized water and then mixing well.

7.2 Prepare 150 μl  working Pre-Amplifier Mix Solution by diluting 0.75 μl Pre-Amplifier Mix 1:200 in 149.25 μl pre-warmed Amplifier Diluent QF. Gently mix the solution and transient centrifuge for 10-20 seconds.

Caution: Amplifier Diluent QF contains formamide, a teratogen, irritant and possible carcinogen. Avoid contact with skin and mucous membranes.

7.3 Remove rubber cement and coverslips from the slide, then put the slide edge on a clean, dry paper towel for 1–2 seconds. Wash slides three times, each with 1 mL/slide of Wash Buffer. Soak sample for 5 minutes during each wash.

  • Critical: During all subsequent washing steps, the slides should be washed fully with wash buffer to eliminate non-specific binding, but do not soak samples in Wash Buffer longer than 30 minutes. Are all the washing steps at room temperature?

7.4 Gently remove the Wash Buffer, put the slide edge on a clean, dry paper towel for 1–2 seconds. Add 150 μl/slide of the working PreAmplifier Mix Solution to the slide, then cover with a 24×50 mm2 coverslip.

7.5 Incubate at 40 °C for 35 minutes in the water bath.

NOTES:

  1. Wash Comp 1 and Wash Comp 2 are provided in the kit, they should be free of precipitates before making a working wash buffer.

 

8. Hybridize with Amplifier

8.1 Prepare 150 μl  working Amplifier Mix Solution by diluting 6 μl Amplifier Mix 1:25 in 144 μl pre-warmed Amplifier Diluent QF. Gently mix the solution and transient centrifuge for 10-20 seconds.

Caution: Amplifier Diluent QF contains formamide, a teratogen, irritant and possible carcinogen. Avoid contact with skin and mucous membranes.

8.2 Remove coverslips from the slide, then put the slide edge on a clean, dry paper towel for 1–2 seconds. Wash slides three times, each with 1 mL/slide of Wash Buffer. Soak sample for 5 minutes during each wash.

8.3 Gently aspirate the Wash Buffer, put the slide edge on a clean, dry paper towel for 1–2 seconds. Add 150 μl /slide of the working Amplifier Mix Solution to the slide, then covered with a 24×50 mm2 Cover Slip.

8.4 Incubate at 40 °C for 35 minutes in the water bath.

 

9. Hybridize with Label Probe

  • Critical: Protect samples from light during all subsequent steps.

9.1 Prepare 150 μl  working Label Probe Mix Solution by diluting 6 μl  Label Probe Mix 1:25 in 144 μl  pre-warmed Label Probe Diluent QF. Gently mix the solution and shortly centrifuge for 10-20 seconds. Protect from direct light exposure.

9.2 Remove coverslips from the slide, then put the slide edge on a clean, dry paper towel for 1–2 seconds. Wash slides three times, each with 1 mL/slide of Wash Buffer. Soak sample for 5 minutes during each wash.

9.3 Gently aspirate the Wash Buffer, put the slide edge on a clean, dry paper towel for 1–2 seconds. Add 150 μl /slide of the working Label Probe Mix Solution to the slide, then cover with a 24×50 mm2 coverslip.

9.4 Incubate at 40 °C for 25 minutes in the water bath.

 

10. Nuclear counterstaining

10.1 Prepare 500 μl  working Hoechst Solution by diluting the 0.5 μl  Hoechst 1:100 in 999.5 μl  PBS. Gently mix the solution. Protect from light.

Note: Nuclear staining can also be done with DAPI. Dilute the 100 X DAPI (provided in the ViewRNA ISH cell assay kit) 1:100 in nuclease free water to obtain a working DAPI solution and perform as follows.

Caution: DAPI is a possible mutagen. Avoid contact with skin and mucous membranes.

10.2 Remove coverslips from the slide, then put the slide edge on a clean, dry paper towel for

1–2 seconds. Wash slides three times, each with 1 mL/slide of Wash Buffer. Soak sample for 5 minutes during each wash.

10.3 Gently aspirate the Wash Buffer, put the slide edge on a clean, dry paper towel for 1–2 seconds. Add 500 μl/slide of the working Hoechst Solution to the slide and incubate for 3 minutes at RT.

10.4 Decant working Hoechst Solution from the slide. Wash slides three times with 1 mL/slide of PBS. Soak sample for 5 minutes during each wash.

10.5 Aspirate PBS and add two drops of fluorescent mounting medium onto the slide, and finally cover with a coverslip to preserve a better fluorescent signal.

10.6 Use a clean paper towel to soak up the excess mounting medium on the sides of the slide when necessary, and seal the slide edges with nail polish.

10.7 Allow the nail polish to dry for at least 15 minutes.

 

 

11. Imaging

A. Samples may be viewed under a fluorescent microscope immediately.

        B. The slide should be stored at 2–8°C protected from light. Fluorescent signals will be stable for up to one week when stored properly.

        C. For viewing signals, use appropriate filter sets. Here we can use filter set Cy3 (550 nm) to detect Probe Set type 1a or use filter set Cy5 (650 nm) to detect Probe Set type 6b.

NOTES:

a. Signal of LP1-550 is visible to unaided eyes under epi-fluorescent microscope, and appear as red dots.

b. Signal of LP6-650 is invisible to unaided eyes under epi-fluorescent microscope and has to be captured by a digital camera.

c. Due to the relative weak signal of FISH, a digital camera with high quantum efficiency is desirable. In addition, high sensitivity mode (e.g. HyD mode in Leica SP8) must be used when using a confocal microscope. The signal is also more prone to be bleached due to the high energy of confocal laser beam. We have also imaged the FISH results using STORM (Type 6 probe only) and STED with some success. Generally, these methods generate higher resolution images but the procedure is much more complex.

d. If one wishes to see colocalization between nucleic acid and protein, a routine immunofluorescence protocol can be performed immediately after 10.2 with no need for fixation or permeabilization step.

 

 

  1. Typical results

Typical imaging results were shown below2. Signal generated by probe set 2 (green) indicates all viral DNA (including core particle DNA and deproteinated DNA), signal generated by probe set 3 (red) indicates deproteinated rcDNA and some core particle DNA in the cytoplasm. Our previous results showed that the rcDNA in HepAD38 has longer plus strand which resulted in a diminished selectivity of probe set 3 toward intranuclear rcDNA and cccDNA. The yellow arrow indicates intranuclear HBV DNA that is detected by both probes. Further differentiation between episomal DNA and integrated HBV DNA is beyond the capability of the assay.

More images are also available at http://www.hepb-atlas.com/home/projects/detail/12

 

A Southern Blot Assay for Detection of HBV cccDNA from Cell Cultures

POSTED ON: 03 Jun, 2019

Cell Cultures

  1. Seed HBV stable cell lines, such as HepG2.2.15 (5), or tetracycline-inducible (tet-off) HepAD38 (6), or tetracycline-inducible (tet-off) HepDE19/DES19 cells (7, 8), in collagen coated 6-well plates at approximately 100% confluence with appropriate culture medium.

Note: Collagen-coated plate is ideal for cells to form confluent monolayers, especially for HepG2-based cells.

Note: HepG2.2.15 cell line is cultured with DMEM/F12 medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin and 100 μg/mL streptomycin, plus 400 μg/mL G418. Tetracycline-inducible (tet-off) cell lines are maintained in the same way as the HepG2.2.15, but with the addition of 1 μg/mL tetracycline.

  1. When the cell monolayer reaches complete confluence (normally 24-48 hours post seeding), change the culture medium with fresh medium. For tetracycline-inducible cell lines, remove the tetracycline from culture medium to induce HBV replication.

Note: To obtain a high level of HBV replication in tetracycline-inducible cell lines, it is critical to withdraw the tetracycline when the cells reach confluence, as HBV replication will arrest cell growth.

Note: HBV infected cells can also be subjected to the following procedures.

 

cccDNA Extraction  from Cell Culture

  1. Lyse cells by adding 1.5 mL of TE buffer (10:10) and 0.1 mL of 10% SDS into each well of a 6-well cell culture plate. Gently mix and incubate the plate for 30 min at room temperature.
  2. Transfer the viscous cell lysate to a 15 mL centrifuge tube. Add 0.4 mL of 5 M NaCl and gently invert the tube 10 times. Let the tube sit at 4°C for at least 16 h to efficiently precipitate proteins and protein-associated DNAs.

Note: Use wide-mouth plastic transfer pipette to transfer the lysate into centrifuge tube.

  1. Centrifuge at 14,500 × g for 30 min at 4°C. Transfer the supernatant to a fresh 15 mL centrifuge tube. To remove the residual protein from the supernatant, add an equal volume of phenol (approx. 2 mL) to the supernatant and mix thoroughly by hand shaking for 10 sec. Centrifuge at 3,500 × g for 10 min at 4°C, and transfer the aqueous phase to a fresh 15 mL tube. Repeat phenol extraction step. Add equal volume of phenol/chloroform (approx. 2 mL) and mix thoroughly by hand shaking, centrifuge at 3,500 × g for 10 min at 4°C and transfer the aqueous phase to a fresh 15 mL tube.

Note: Use leak-proof tube with screw cap. Do not shake the tube too vigorously or too long, which it may physically nick the supercoiled cccDNA.

  1. Add two volumes of 100% ethanol (approx. 4 mL) and mix thoroughly by inverting the tube several times. Incubate at room temperature overnight to precipitate DNA.

Note: Do not incubate at low temperature. Because high concentration of salt is used in Hirt extraction, incubation of DNA solution at low temperature will precipitate high amount of salt.

  1. On the third day, centrifuge the tube at 3,500× g for 30 min at 4°C, and discard the supernatant. Add an equal volume of 75% ethanol (approx. 2 mL) and gently flick the tube to wash the DNA pellet. Centrifuge at 3,500× g for 15 min at 4°C.
  2. Discard the supernatant. Allow the pellet to air dry for about 10 min at room temperature. Dissolve the DNA pellet in 25 μL TE buffer (10:1).

 

Agarose Gel Electrophoresis

  1. Prepare 150 mL of 1.2% gel by mixing 1.8 g agarose, 5 mL of 30× TAE buffer, and 145 mL of distilled water in a microwavable flask. Microwave the flask until the agarose is thoroughly dissolved. Let it cool down to about 50°C at room temperature. Pour the liquid gel into the gel tray (12cm×10cm) with an appropriate comb inserted. Let the gel solidify at room temperature before removal of the comb.
  2. Prepare 600 mL of gel running buffer by diluting 20 mL of 30× TAE buffer with de-ionized water.
  3. Prepare samples by adding approximately 3 μL of 10× loading buffer to 25 μL of each sample. Pipette up and down to mix well.
  4. Assemble submarine gel running unit by correctly positioning the gel box, adding running buffer into the chamber, and loading 28 μL of each sample into a separate well. Connect the gel running unit to a power supply and run gel at 25 V for overnight.

 

Southern Blot

  1. Disconnect the gel running unit and take out the gel carefully.
  2. Submerge the gel in a tray containing freshly prepared 0.2 M HCl. Agitate gently for 10 min at room temperature to depurinate the DNA samples from the gel. The bromophenol blue dye in the gel should gradually turn grey during this process.
  3. Rinse the gel three times with distilled water.
  4. Submerge the gel in a tray of denaturing buffer, and agitate gently for 1 h at room temperature. The loading dye in the gel should turn back to blue during this process.
  5. Rinse the gel three times with distilled water.
  6. Submerge the gel in a tray of neutralization buffer, and agitate gently for at least 1 h at room temperature.
  7. Soak the Hybond-XL membrane (12×10cm) in a tray with de-ionized water for 5 min at room temperature. Replace the water with 20× SSC and continue to agitate for 15 min.
  8. To transfer DNA from the gel to the Hybond-XL membrane, assemble the transfer apparatus as following: layer two sheets of Whatman 3 MM filter paper in the gel transfer tray and pour 20× SSC transfer buffer on top to completely wet the Whatman papers. Smooth out the bubbles in between the Whatman papers and the tray gently by a plastic roller, and get rid of the excess amount of the transfer buffer. Place the gel on top of the Whatman papers with the side containing the loading wells facing down; make sure there is no air bubble between the Whatman papers and the gel. Place the presoaked Hybond-XL membrane on top of the gel and use a plastic roller to get rid of the air bubbles between the gel and the membrane. Layer two sheets of 20× SSC pre-wet Whatman papers (12×10cm) on top of the membrane, use plastic roller to remove air bubbles in between. Place a stack of dry paper towels (about 4 inches) pre-cut to the gel size on top of the Whatman papers. Seal the transfer tray with plastic wrap to prevent buffer evaporation during transfer. Put some weight (approx. 500g. such as metal plate, casserole dish etc.) on top of the transfer apparatus (Fig.1). Let the assembled transfer apparatus sit still on a flat surface and transfer for 24-48 h.

Note: When assemble the Southern blot transfer apparatus, avoid possible short-circuits of capillary liquid flow. The DNA will not be efficiently transferred if the blot short circuits. Use parafilm strips to seal the edges of the gel.

  1. After gel transfer, disassemble the blot apparatus and flip over the membrane with the gel still attached, and mark the position of each loading well on the membrane by a pencil. Wrap up the gel and paper towels by plastic wrap and discard.

Note: After transfer, the gel becomes thinner. Use the pencil tip to penetrate the loading wells and mark their positions on the membrane. This step is for labeling the DNA binding site of the membrane and tracking the running lanes with orientation.

  1. Crosslink the Hybond-XL membrane in a UV crosslinker chamber with UV energy dosage at 120 mJ/cm2. The membrane can be directly subject to hybridization, or sandwiched between two sheets of Whatman filters and stored at -20°C.

 

Probe Preparation for HBV DNA Detection

  1. Prepare rNTP (supplied with Riboprobe® system) solution by mixing 50 μL of each 10 mM rATP, 10 mM rGTP, 10 mM rCTP, and 0.2 mM UTP.
  2. Add 4 μL of 5 × reaction buffer, 4 μL of rNTP solution prepared from the previous step, 2 μL of 100 mM DTT, 0.25 μg of SalI-linearized pGEM-HBV DNA template, 1 μL of RNasin and 1 μL of SP6 polymerase into a nuclease free tube. Adjust the volume to 14 μL with nuclease-free water before adding 6 μL of [α-32P] UTP into the mixture. Incubate the reaction at 37ºC for 1 h.

Note: Linearization of the plasmid pGEM-HBV by SalI is an optimal step to produce “run-off” transcripts derived from the HBV sequence only. Other restriction enzyme, which singly cuts the vector sequences downstream of SP6-HBV transcription cassette but does not leave a 3’ overhang, can also be used.

Note: All radioactive materials and procedures should be handled strictly by following the institutional laboratory safety regulation rules.

  1. To remove the DNA template, add 1 μL of RNase-free RQ1 DNase to the reaction and incubate at 37°C for 15 min.
  2. Add 15 μL of 5M NH4OAc to stop the reaction. To precipitate the RNA probe, add 113 μL of nuclease-free water, 4 μL of yeast RNA, and 150 μL of isopropanol, gently mix and incubate at room temperature for 10 min.
  3. Centrifuge the mixture at 12,000× g for 30 min at 4°C, and discard the supernatant carefully without disturbing the pellet. Dissolve the probes in 400 μL of de-ionized formamide, followed by measurement of the counts per minute (CPM) of acid insoluble 32P with scintillation counter (PerkinElmer). Store the probes at -20°C.

Note: 32P-radiolabeled HBV DNA probes prepared by random priming or end labeling can also be used in the hybridization.

 

Hybridization

  1. Place the crosslinked membrane in a hybridization tube with the DNA-binding side facing the center of the tube. Add 5 mL of EKONO™ hybridization buffer, and pre-hybridize the membrane by rotating the hybridization tube at 65°C for 1 h in a hybridization oven.

Note: If using other commercial hybridization buffers, follow the pre-hybridization and hybridization conditions recommended by the manufacturers.

  1. Replace the pre-hybridization buffer with 5 mL of fresh EKONO™ hybridization buffer, and add HBV riboprobes with 1×107 Rotate the hybridization tube at 65°C overnight.
  2. Discard the hybridization solution on the following day, and wash the hybridization membrane with approximately half a tube of wash buffer. Rotate at 65°C for 30 min.
  3. Discard the wash buffer, and replace with half a tube of fresh wash buffer. Continue to wash at 65°C for 1 h.
  4. After the second wash, take out the membrane and dry it with paper towels, making sure the membrane is not cracked or wrinkled during this process. Seal the membrane with plastic wrap. Place the membrane in a phosphorimager cassette with the DNA-binding side facing up. Layer the phosphor imaging screen on top facing the DNA-binding side of the membrane. Close the cassette tightly and expose overnight in the dark.
  5. Scan the phosphor imaging screen with phosphorimager system. Store the membrane properly and erase the signal in the phosphor imaging screen with intense light for re-use.
  6. A typical phosphor image of HBV DNA Southern blot is shown in Fig. 2 (also see in references (7, 9)). Quantify the signal intensity of the cccDNA bands with software provided by the phosphorimager system.

 

Figure 1:  Assembly of HBV DNA Southern blot transfer apparatus. See text for details.

Figure 2:  Detection of HBV DNA by a Southern blot assay. A typical pattern of HBV cytoplasmic core DNA (lane 2) and Hirt DNA (lane 3) upon Southern blot hybridization is shown. RC: relaxed circular DNA; DP-RC: deproteinized RC DNA; CCC: covalently closed circular DNA; SS: single-stranded (-) DNA. M: HBV genomic-length DNA marker. The approximate size of each HBV DNA species is labeled on the left. To further validate the authenticity of HBV cccDNA, the Hirt DNA sample in lane 3 has been heated to 85°C for 5 min before gel loading, a condition that denatures DP-rcDNA into SS DNA, while the cccDNA stays undenatured and its electrophoretic mobility remains unchanged (lane 4). The DNA sample from lane 4 is further digested with EcoRI, in which condition the cccDNA is linearized to a genome-length double-stranded DNA (lane 5). Reproduced from (9) with permission from Elsevier.

Stem Cell Derived Hepatocyte-Like Cells for Hepatitis B Virus Infection

POSTED ON: 30 Apr, 2019
  1. Stem cells culture and passage

Routine non-colony-type culture of human pluripotent stem cells (hPSCs)

–       Colony type cultures of hPSCs (hESC or hiPSC) were adapted to non-colony monolayer type culture as described previously by Chen et al. (Chen KG, et al. Stem Cell Research 2012).

–       Once adapted to non-colony type culture, hPSCs are routinely maintained in 6-well plate containing 2 ml of mTeSR1, previously coated with high-concentrated growth factors reduced (GF (-)) Matrigel (0.4 mg/ml).

–       On the day cells reach confluence, new plates are coated with high concentrated GF (-) Matrigel.

–       250 µl of GF (-) Matrigel are resuspended in 6 mL of DMEM-F12 (Final concentration: 0.4 mg/ml), and allowed to rest for 1 hour on ice for homogenization.

–       1 mL of the high concentrated GF (-) Matrigel solution is transferred to each well of a 6 well plate.

–       The plate is incubated at 37°C for 15 minutes and then sealed for 4°C storage.

–       The day after, polymerized Matrigel should be visible at high magnification (x40) using a contrast phase microscope.

–       The overconfluent hPSCs, ready for passage, are washed once with PBS.

–       Cells are incubated with 1 mL of gentle dissociation buffer (Accutase, Life Technologies) at 37°C for 5-8 minutes.

–       Cells are resuspended in 9 mL of mTeSR1 medium.

–       At this point, 50 µL of cell suspension can be collected for cell count.

–       Centrifugation 3 minutes at 2,000 rpm at RT

–       Supernatant is discarded.

–       The pellet is resuspended in mTeSR1.

–       Cells are passed 1/4 to 1/5 on high-concentrated GF (-) Matrigel-coated plates in mTeSR1 + 10 µM of Rock Inhibitor.

–       Cells are cultured at 37°C, 5% CO2, normal O2, with mTeSR1 medium changed daily.

 

Note: Different clones of human stem cells (either iPSC or ESC) may differentiate differently to hepatocyte-like cells, which may also have different infectious efficiency by HBV. It is recommended to test various human stem cell lines to obtain the optimal lines for differentiation and infection. See attached references for lines we have used successfully for this purpose. We will be depositing our stem cell clones to the HBV Resource Repository.

 

  1. Hepatocyte-like cells differentiation

Passing hPSCs for differentiation (timing: 1 day)

Day -1: Passage hPSCs in 6 well plate for definitive endoderm induction

–       Pre-coating of 6 well plate with low concentration GF (-) Matrigel: 250 µL of GF (-) Matrigel (Corning) in 20 mL of DMEM-F12 (0.125 mg/mL)

–       Allow to rest 1 hour on ice to ensure homogenization.

–       Distribute 1 mL per well of a 6 well plate.

–       Incubate at least 15 minutes at 37°C for polymerization.

–       hESCs or hiPSCs are washed with 2 mL PBS at RT.

–       Add 1 mL of gentle dissociation buffer (Accutase, Life Technologies) per well, 5-8 minutes at 37°C.

–       Resuspend in 9 mL of mTeSR1.

–       Count cells with trypan blue exclusion test.

–       Centrifuge 3 minutes at 2000 rpm at RT.

–       Discard supernatant.

–       Resuspend cell pellet at a concentration of 1 millions cells per mL + 10µM of Rock Inhibitor.

–       Transfer 2 mL (2 millions cells) per well of a 6-well plate, previously coated with low concentrated GF (-) Matrigel.

–       Incubate the cells at 37°C, 5% CO2, normal O2, overnight.

 

Definitive endoderm induction (timing: 4 days)

Day 0: Definitive endoderm induction (day 1, D1) Supplement MR and CJ

–       Cells seeded the day before should cover more than 90% of the well.

–       1 wash with PBS at RT to discard dead floating cells.

–       Add 2 mL of STEMCELL Technologies Definitive Endoderm Basal Medium + 20 µL of supplement MR + 20 µL of supplement CJ per well.

–       Incubate overnight at 37°C, 5% CO2, normal O2.

 

Trouble shooting:

–       Cells are not confluent: Seed 2 million living cells at day 0, in presence of 10 Mm Rock Inhibitor.  Ensure high quality of pluripotent stem cells.

 

Day 1: Definitive endoderm induction (D2) Supplement CJ

–       Gently shake the plate to float dead cells and discard medium.

–       Add 2 mL of Basal Medium + 20 µL of supplement CJ per well.

–       Incubate at 37°C, 5% CO2, normal O2.

 

Day 2: Definitive endoderm induction (D3) Supplement CJ

–       Discard medium.

–       Add 2 mL of Basal Medium + 20 µl of supplement CJ per well.

–       Incubate at 37°C, 5% CO2, normal O2.

 

Day 3: Definitive endoderm induction (D4) Supplement CJ

–       Discard medium.

–       Add 2 mL of Basal Medium + 20 µl of supplement CJ per well

–       Incubate at 37°C, 5% CO2, normal O2.

 

Day 4: Assessment of definitive endoderm (=DE) induction

–       At that point, cells should cover the entire surface of the wells, between 3 and 3.5 millions cells per well of a 6 well plate.

–       DE cells can be assessed for expression of definitive markers SOX17 and FoxA2, by immunofluorescence(IF) (Fixation with PFA 4%) or FACS analysis (after resuspension with Accutase).

 

Trouble shooting:

–       Cells are not confluent: May indicate higher susceptibility to cell death during supplement MR treatment.

–       Cells are negative for SOX17/FoxA2: Validate quality of stem cell population used (IF for pluripotency markers).

 

Hepatic specification (timing: 8 days)

Day 4: Passage for Hepatic Specification

–       Plates used for the hepatic differentiation must be coated with low concentration GF (-) Matrigel (0.125 mg/mL) as described on day -1

Format                    Surface            Volume of Matrigel per well

6-well plates:         9.5 cm2           1 mL

12-well plates:                3.8 cm2           0.5 mL

24-well plates:                1.9 cm2           0.25 mL

96-well plates        0.32 cm2 60 µL

384-well plates    0.056 cm2       40 µL

 

–       Discard supernatant.

–       Wash cells once with RT PBS.

–       Add 1 mL of Accutase per well.

–       Incubate 5-8 minutes at 37°C.

–       Resuspend in 9 mL of Differentiation Medium.

 

Differentiation Medium:

DMEM (4.5 g/L glucose)                        225 mL

F12 medium                            225 mL

Knock Out Serum Replacement           50 mL

NEAA                                        5 mL

Penicillin Streptomycin                 5 mL

Glutamine                               5 mL

 

–       Use 50 µL for cell count with trypan blue exclusion test.

–       Centrifuge at 2000 rpm, 3 minutes at RT

–       Supernatant is discarded.

–       Cell pellet is resuspended in differentiation medium + 100 ng/mL of HGF + 1% DMSO + 10 µM Rock Inhibitor, at a concentration depending on the well format chosen for subsequent hepatic specification.

–       The cell suspension is then distributed in the well pre-coated with low concentration GF (-) Matrigel.

–       Concentration of cells for hepatic specification can vary from cell line to cell line, but we suggest a starting concentration of 79.000 cells/cm2.

 

Format                    Surface   Suggested concentration      Volume per well

6 well plates:          9.5 cm2               750.000 cells per well          2 mL

12 wells plates:              3.8 cm2            300.000 cells per well             1 mL

24 wells plates:              1.9 cm2         150.000 cells per well            0.5 mL

96 wells plates               0.32 cm2 25.000 cells per well              100 µL

384 wells plates    0.056 cm2       4.400 cells per well                40 µL

 

–       Cells are then incubated overnight at 37°C, 5% CO2, normal O2.

 

Day 5: Hepatic Specification Day 2

–       At that time, most of the cells should be adherent, covering around 75% of the well surface.

–       Cells are washed once with PBS.

–       Add 2 mL of Differentiation medium + 100 ng/mL HGF + 1% DMSO per well.

–       Incubate the cells at 37°C, 5% CO2, normal O2.

 

Trouble shooting:

        Cells are non-adherent: On day 4, ensure the extracellular matrix is polymerized.  Polymerized Matrigel should be visible using an inverted contrast phase microscope at high magnification (>20x).  Do not forget Rock Inhibitor at the time of seeding.

 

Day 6-7 : Hepatic Specification Day 3-4

–       Daily replace the Differentiation medium + 100 ng/mL HGF + 1% DMSO

–       Incubate the cells at 37°C, 5% CO2, normal O2.

 

 

Day 8-11: Hepatic Specification Day 5-8

–       At that time, the cells should be confluent, and be growth-arrested.

–       Daily replace the Differentiation medium + 100 ng/mL HGF + 1% DMSO

–       Incubate the cells at 37°C, 5% CO2, normal O2.

 

Day 12: Assessment Hepatic specification

–       Cells at day 12 can be assessed for expression of hepatoblast markers, like the Hepatocyte Nuclear Factor 4 alpha (HNF4A), a master regulator of hepatic differentiation, and alpha-fetoprotein (AFP), by immunofluorescence or FACS analysis.

–       AFP secretion can be assessed by ELISA on supernatant of culture collected on day 12.

 

Trouble shooting:

–       Cells are not confluent: Seed more cells on day 4

–       Cell population is very heterogeneous: validate quality/homogeneity of the DE cell population before plating.

–       Cells are negative for AFP: Check quality/lot/concentration of HGF.

 

Hepatic maturation (timing: 3 days)

Day 12-14: Hepatic Maturation Day 1-3

–       Supernatant is discarded.

–       Daily replace by Differentiation medium + 10-7 M Dexamethasone.

 

Day 15: Assessment of Hepatic Maturation

–       At that time, the cell morphology should be similar or close to the one of PHHs: Polygonal cells, tight junctions, small round nuclei, sometimes binucleated cells, and lipid droplets visible in the cytoplasm.

–       Expression of hepatic marker, for example albumin (ALB) , alpha-fetoprotein (AFP) , alpha-1-antitrypsin (AAT) can be assessed by IFA.  Secretion of AFP or ALB can also be assessed by ELISA on supernatant of differentiated cells.

       

Trouble shooting:

–       Cells not confluent at day 15: Seed more cells on day 4.  Check that cells were seeded in presence of Rock Inhibitor.  Monitor cell death (floating cells) to ensure viability of the cells.

–       Cells too confluent / multilayered at day 15: Ensure the quality/homogeneity of the DE cells, as the DE-derived hepatoblasts should be growth-arrested.  Cells on the top layer are usually non-directed differentiation from non-DE cells cultured in presence of DMSO. Seed less DE cells per cm2 on day 4.

–       Cells are not polygonal: Assess expression of AFP to validate the hepatic specification.  If cells are still AFP positive, it means the hepatic maturation didn’t occur.  Check quality/concentration/lot of dexamethasone.

–       Cells are negative for ALB but positive for AFP/HNF4A: Check dexamethasone, ensure good cell density.  If cells not confluent, seed more cells on day 4.

 

  1. HBV infection

Cell culture derived HBV was concentrated from the supernatant of HepG2.2.15 cells using centrifugal filter devices (Centricon Plus-70, Biomax 100.000, Millipore Corp., Bedford, MA) and titered by HBV DNA qPCR. Immediately after collection, the virus stock was divided in aliquots and stored at -80°C until use. For infection, inoculation of cells was performed with multiplicity of infection (MOI) 200-300 in WEM medium containing 5% PEG 8000 (Sigma Aldrich, St. Louis, USA) for 16 h. At the end of incubation period, HLCs were washed three times with PBS and cultured in WEM medium.

 

MAINTENANCE OF HLCs (TIMING: 0-3 WEEKS)

At that point, differentiated HLCs can be kept more than 2 weeks in the Hepatocyte Maintenance Medium.

 

Hepatocytes Maintenance Medium:

William’s E Medium              500 mL

FBS                           50 mL

Penicillin Streptomycin         5 mL

Human insulin                       1 µg/mL (350µL)

Hydrocortisone                     5 µg/mL

DMSO                              1.8%

 

The medium should be changed every 2-3 days.

At that stage, the cell morphology should continue toward a more mature hepatic phenotype.

 

Trouble shooting:

–       Cell death: Ensure the right concentration of DMSO, as too high concentration may exert a cytotoxic effect on the cells.

Detection and characterisation of integrated Hepatitis B virus DNA using inverse nested PCR

POSTED ON: 20 Apr, 2019

This method has previously been described in greater detail as a chapter in the book Methods in Molecular Biology: Hepatitis B Virus (7) and as an online open-access video in the Journal of Visualised Experiments (10). The procedure is summarized in Figure 1.

1A. Extraction of DNA (liver tissue from HBV-infected patients)

  • Within a biosafety cabinet, use a sterile plastic Petri dish and a sterile scalpel blade to excise a ~5 mg fragment from a snap-frozen sample of biopsy or autopsy liver tissue.
  • Place the tissue immediately in a 2 mL screw-cap Eppendorf tube with 400 μL of digestion solution (100 mM NaCl, 0.5% SDS, 50 mM Tris pH 7.5, 10 mM EDTA, 2 mg/mL proteinase K).
  • Incubate in a thermomixer rotating at 3,000 rpm at 55°C for >2 hr (extendable to overnight) until no obvious pieces of liver parenchyma are visible. Some pieces of fibrotic tissue or fat may still be obvious.
  • In a fume cupboard, add 400 μL of UltraPure Phenol and mix by inversion.
  • Centrifuge at 14,000 g for 10 min and transfer the top aqueous layer to a clean 2 mL screw-cap Eppendorf tube, taking care to avoid any milky or cloudy interface.
  • Repeat steps 4-5 twice with 400 μL of 25:24:1 UltraPure phenol:chloroform:isoamyl alcohol in the place of the phenol.
  • Add 35 μL of 3 M sodium acetate pH 4.6 to the extracted DNA, followed by of 800 μL of AR grade ethanol.
  • Incubate at -20°C overnight or at -80°C for 4 hr to precipitate the DNA.
  • Pellet the precipitated DNA by centrifugation at 14,000 g.
  • Wash the pellet with 1 mL of 70% ethanol.
  • Repeat Step 10.
  • Vacuum-dry for 20 min and redissolve in 50 μL of DNase-free water or 100 mM Tris-HCl pH 7.5.
  • Estimate the final DNA concentration and purity using spectrophotometry.

 

1B. Extraction of DNA (after in vitro infection of cultured cells)

  • Maintain cultured Huh7-NTCP cells (11, 12) in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% v/v fetal bovine serum, 1 x Penicillin/Streptomycin, and 2 mM L-glutamine. This has previously been reported in detail (13).
  • Seed Huh7-NTCP cells into a 12-well plate with 1 mL of DMEM containing 2×105 cells/mL.
  • If testing cell treatments (e.g. HBV inhibitors), apply these to the culture supernatant 4 hours after seeding (1 day prior to HBV infection). For a negative control, use 200nM Myrcludex B (a potent HBV entry inhibitor (14)).
  • Use heparin-column purified (15) supernatant from HepAD38 (16) as an inoculum to infect cells at 1000 VGE/cell in 500 µL of culture media (DMEM supplemented with 10% v/v fetal bovine serum, 1 x Penicillin/Streptomycin, 2 mM L-glutamine, and 2.5% v/v DMSO), containing 4% w/v polyethylene glycol 8000 (dissolved in 1x phosphate-buffered saline; PBS).
  • Culture cells in a 37°C incubator (set at 5% CO2 and 90% humidity) overnight.
  • Wash twice with 1 mL sterile 1x PBS at 16-24 hours post-infection.
  • Replace culture media every two days following HBV infection until harvest.
  • At day 3 post-infection, treat cells with 5 µM Tenofovir disoproxil and 10 µM Lamivudine to limit production of HBV replicative intermediates that are amplifiable by invPCR.
  • At day 5 post-infection, trypsinise cells with 200 µL Trypsin-EDTA and resuspend them in 2 mL of DMEM (supplemented with 10% v/v fetal bovine serum, 1x Penicillin/Streptomycin, and 2mM L-glutamine), containing 5 µM Tenofovir disoproxil and 10 µM Lamivudine.
  • Transfer the cell suspensions to a 6-well plate to induce one round of mitosis, which has been reported to induce loss of HBV cccDNA (17) and other HBV DNA intermediates that are amplifiable by invPCR.
  • At day 7 post-infection, trypsinise the expanded cells in 400 µL Trypsin-EDTA and resuspend the mixture in 1 mL DMEM.
  • Place the suspension in a 1.5 mL Eppendorf tube, pellet cells by centrifugation at 500 x g for 5 minutes, and remove supernatant by aspiration.
  • (Optional) Store cell pellets at -20°C until ready for DNA extraction.
  • Extract DNA from the cell pellet as above, or by using a column-based DNA extraction kit as per the manufacturer’s instructions.
  • Estimate the final DNA concentration and purity using spectrophotometry. The DNA yield in a 100 μL elution volume is generally ~250-400 ng/μL.

 

  1. Inversion of DNA
    • Aliquot ~1.5-2.5 μg of the total DNA extract into a 200 μL PCR tube.
    • Add the appropriate amount of restriction enzyme master-mix to result in a 40μL reaction volume, containing 1x CutSmart buffer and 10U NcoI-HF.
    • Mix reactions thoroughly and spin down in a small tube centrifuge.
    • Incubate the restriction enzyme reaction in a PCR machine at 37°C for 1 hour for optimal digestion efficiency.
    • Inactivate the restriction enzyme by incubating at 80°C for 20 minutes.
    • Transfer the entire restriction enzyme reaction into a 1.5 mL Eppendorf tube.
    • Add 400 μL of 1x T4 DNA ligase buffer and 500 U T4 DNA ligase and mix thoroughly. The large reaction volume encourages intra-molecular (as opposed to inter-molecular) ligation of the digested DNA fragments.
    • Incubate the ligation reaction at room temperature for 2 hours to ensure complete ligation.
    • Inactivate the T4 DNA ligase at 70°C for 20 minutes.
    • Add 10 μL of 10% w/v sodium dodecyl sulphate to ensure complete inactivation of the T4 ligase.
    • Mix the tubes by pulse vortexing and briefly spin down the reaction mix.
    • Add NaCl to a final concentration of 100 mM and dextran (35 -45 kDa) to a final concentration of 90 µg/mL.
    • Mix the tubes by pulse vortexing and briefly spin down the reaction mix.
    • Add 900μL of 100% ethanol and mix by inversion.
    • Precipitate the DNA at -20°C overnight.
    • Pellet the precipitated DNA by centrifugation at 14,000 x g for 15 minutes.
    • Wash the pellet with 500 μL of 70% v/v Ethanol and centrifugation at 14,000 x g for another 15 minutes.
    • Remove the ethanol by aspiration with a P200 pipette.
    • Air-dry the DNA pellet at RT for 20 minutes.
    • Redissolve the pellet in 20 μL H2
    • Add 20 μL of a restriction enzyme master-mix to result in a 40 μL reaction volume, containing 1x CutSmart buffer and 5U BsiHKAI and 5U SphI-HF.
    • Incubate the restriction enzyme reaction in a heat-block at 37°C (the optimal temperature for SphI-HF) for 1 hr.
    • Incubate the restriction enzyme reaction in a heat-block at 65°C (the optimal temperature for BsiHKAI) for 1 hr.
    • Store the inverted DNA at -20°C until required.

 

  1. Nested PCR
    • Prepare 1 mL of 1x Amplitaq Gold PCR mix containing the outer forward and reverse primers at a concentration of 0.5 µM.
    • Add 170 μL of the 1x Amplitaq Gold PCR mix to wells A1 and E1 of a 96-well PCR plate.
    • Add 120 μL of the 1x Amplitaq Gold PCR mix to wells B1 to H1.
    • For cell culture samples, as significant clonal proliferation has not occurred, 2 different inverted samples can generally be analysed on the same PCR plate. In this case, add 10 μL of the inverted DNA to wells A1 and E1. For liver tissue samples, use only one DNA extract per plate and add inverted DNA to only well A1.
    • Mix the reaction in each well by gently pipetting ~10 times using a P1000 set at 100 μL.
    • Serially dilute samples from wells A1 to D1 (or A1 to H1 for tissue samples) at a ratio of 1:3 by transferring 60 μL at each step. Mix at each step by gently pipetting ~10 times using a P1000 set at 100 μL. Avoid forming bubbles.
    • Repeat Step 3.3 for well E1, diluting down to well H1.
    • Aliquot 10 μL of the reaction mixture using a multi-channel pipette from wells A1-H1 into wells A2-H2, A3-H3, and so forth until wells A12-H12 of the 96-well plate.
    • Remove potential amplicons from a reusable silicon mat seal using DNAZap, rinse the mat thoroughly with DNA-free water and air-dry.
    • Cover the PCR plate with the dry silicon mat, pressing firmly.
    • Place the plate in a PCR machine and run the following program: 10 min at 95°C; 35 cycles of 15 sec at 95°C, 15 sec at 54°C and 3 min at 72°C; 7 min at 72°C, then hold at room temperature.
    • Carefully remove the silicon mat to avoid cross-contamination between wells.
    • Heat the pins of a 96-pin replicator to red-hot with a Bunsen burner and then cool for at least 5 minutes at room-temperature.
    • Fill the wells of a second PCR plate with 10 μL of GoTaq Flexi Green PCR mix and the inner forward and reverse primers at 0.5 µM.
    • Use the cooled replicator to transfer PCR products of the first 96-well plate to a second 96-well plate.
    • Carry out the nested PCR using the same conditions as Step 3.10, except for changing the initial denaturation step at 95°C from 10 minutes to 2 minutes.

 

  1. PCR product isolation and gel extraction
    • Analyse the PCR products by gel electrophoresis using a 96-well 1.3% w/v agarose gel. For a 100mL agarose gel, run at 200V for 10-15 minutes.
    • Excise the DNA bands from agarose gels using disposable drinking straws. It is sufficient to isolate bands only from those dilutions where single PCR products can be resolved.
    • For each PCR product, place the straw and agarose gel plug into a 1.5mL Eppendorf tube and then trim the straw to size with scissors.
    • (Optional) Store at tubes -20°C for later extraction.
    • Squeeze each straw to liberate the agarose plug into each Eppendorf tube.
    • Add 300 μL of QX1 Buffer (Qiagen), 5 μL of QiaexII glass beads (Qiagen) to each tube.
    • Extract the PCR products as per manufacturer’s instructions for the QiaexII gel extraction kit (except using half volumes for washing steps) and elute DNA from the beads with 30 μL of water or Elution Buffer.
    • Submit purified DNA for Sanger sequencing using inner forward primer used in the nested PCR.
    • Confirm virus-cell DNA junctions by nucleotide BLAST analysis (using default settings aligning to the entire nucleotide collection). Trim the 5’ HBV DNA sequence before re-running analysis if only partial alignment of the sequence is observed.
    • Calculate the integration frequency by multiplying the dilution factor of inverted DNA templates by the number of virus-cell junctions detected at that dilution, followed by normalisation to the amount of total DNA input into the inversion reaction. Generally, the integration frequency is on the order of 1 in ~104 

Table 1. PCR primers used in invPCR, reproduced from (6).

PCR Forward Primer Sequence

(5’ -> 3’)+

Position on HBV genome* Reverse Primer Sequence

(5’ -> 3’)+

Position on HBV genome*
Outer TTCGCTTCACCTCTGCACG 1603-1621 AAAGGACGTCCCGCGCAG 1422-1405
AAAGGACGTCCCGCGAAG 1422-1405
Inner TGGAGACCACCGTGAACG 1626-1643 AGTACAGCCTAGCAGCCAT 1388-1370
CGCATGGAGACCACCGTGA# 1623-1641 CACACCCTAGCAGCCATGG 1390-1372
CACAGCCTAGCAGCCATGG# 1390-1372
CGCATGGAAACCACCGTGA 1623-1640 CACAGCCTAGCAGCCATGG 1390-1372

+ These outer and inner PCR primer sets were previously used by us to amplify VCJ by nested PCR. A single forward primer could be used with one of multiple reverse primers. The specific primer set used for any given patient was based on their compatibility with the DNA sequence of the infecting HBV DNA strain.

* Primer positions were based on numbering of the HBV DNA nucleotide sequence from NCBI Reference Sequence: NC_003977.2.

# These primers are compatible with the Genotype D ayw strain (NCBI Reference Sequence: NC_003977.2) used in the majority of laboratory experiments.

Tips

  • As this is a highly sensitive technique that amplifies down to single copies of DNA template and uses the same PCR primers for separate samples, limiting DNA contamination is a major issue. General strategies to limit PCR contamination include:
  • Identify and establish 4 physically separate areas including (from “more dirty” to “less dirty”):
  1. Tissue DNA extraction area (carried out in a biosafety cabinet with a UV lamp for decontamination).
  2. DNA extraction (pre- and post-PCR), inversion and sequencing reaction set-up area;
  3. Template addition to PCR and flamed-pin transfer area (we have used PCR hoods with a decontaminating UV lamp with good results);
  4. Stock solution and PCR master-mix set-up area to be used to prepare fresh dedicated reagents for carrying out the inversion reaction, PCR, and DNA extractions. Make up buffers from new powdered stocks.
  • Have separate lab gowns (which are changed often) for all 4 areas and change gloves when moving from “more dirty” to “less dirty” areas.
  • Limit cross-current air flow within the lab, especially in the “template addition to PCR” area. Cross-contamination of wells at this point will lead to inaccurate quantification of virus cell junctions (VCJ). To locate an area of the lab with the least cross-currents, we hold up a piece of tissue and measure the amount of air movement by eye. PCR hoods can also be used to limit these currents. Use negative control wells in the 96-well PCR to test for cross-contamination.
  • Decontaminate all work surfaces with 0.5% sodium hypochlorite solution or DNA-Zap. Wipe off with a damp paper towel, and then dry with a clean paper towel.
  • Never decontaminate with 70% ethanol for the purpose of removing DNA contaminants. Ethanol will fix any DNA to the surface, making it even harder to remove.
  • To design a new inversion protocol, our suggested criteria for invPCR designs are as follows:
    1. As a guideline restriction enzyme 1, 2 and 3 sites (see Figure 1) should occur within ~1 kbp of the expected VCJ site on the HBV genome at ~nt 1832 (1, 18) to limit the size, and so improve circularisation and PCR amplification of the excised fragments.
    2. The design must allow >40 nt between the RE2 restriction site and the expected VCJ site to allow nested primer design.
    3. Similarly, the design must allow >40 nt between the RE1 and RE2 sites on the HBV genome for nested primer design.
    4. RE1 site should occur as frequently as possible in the host genome, but occur very rarely (preferably uniquely) in the HBV DNA genome to limit formation of amplifiable cccDNA-derived fragments.
    5. RE1 should be able to be heat-inactivated to simplify transition to the ligation step.
    6. RE2 and particularly RE3 should occur as frequently as possible in the HBV genome to limit formation of amplifiable cccDNA-derived fragments, but occur rarely in the human genome to avoid cleavage between the VCJ and a downstream RE1 site, resulting in an unamplifiable fragment.
  • To keep the liver tissue cool during sectioning prior to DNA extraction, we place an inverted plastic petri dish or lid on top of dry ice. We then put the frozen tissue in the inverted lid and break into 5 mg fragments (we generally analyse five 5 mg liver fragments per patient), changing dishes and scalpels for each patient. If forceps are used during the extraction procedure, immerse in 0.5% sodium hypochlorite solution for at least 15 min between samples, rinse in water and dry on paper towels.
  • Using the inversion and PCR amplification conditions described, full-length cccDNA should not amplify to detectable levels. However, it may still be necessary to purify high molecular weight DNA to reduce cccDNA contamination, since residual amplification may result due to 1) naturally occurring deletion mutants of cccDNA, or 2) low level star activity of RE1 and circularisation of cccDNA fragments. In each case, the DNA molecules generated are small enough to be amplified to detectable levels. An additional gel purification step using 1% low-melting temperature agarose (Bio-Rad) can be done before the inversion step to isolate high-molecular weight cellular DNA from low (<3.2 kb) molecular weight intermediates. It is important to quantify the DNA before and after this step to account for any lost DNA yield during the purification in order to calculate VCJ number in the original DNA sample.
  • As a lower concentration of DNA should improve the inversion efficiency, less DNA can be put into the assay. The amount of DNA can be determined by probability calculations of DNA fragment self-ligation as described below.
  • The large 400 μL ligation reaction volume favours self-ligation and circularisation of DNA fragments as opposed to intramolecular ligation. The probability of circularization of linear DNA during ligation is governed by the ratio of factors j and i (19),where j is the effective concentration of one end of a DNA molecule in the neighbourhood of the other end of the same molecule in ends/mL and i is the total concentration of DNA ends within a given solution in ends/mL. The probability of circularisation can be shown as simply j/i. When j/i = 1, equal products of circular and linear ligations are expected. When j/i > 1, self-ligated circular forms are favoured and when j/i < 1, intermolecular linear ligation is preferred. Covalently closed circular structures of DNA can be found only when j/i < 2-3 (19). Using the assumptions that:
  1. RE1-cleaved VCJ-containing fragments are 1000 bp long;
  2. 75 μg of DNA in the ligation reaction (over-estimate);
  3. Cellular DNA has a random distribution of bases ;
  4. RE1 has a 4 base recognition sequence; and
  5. 400 uL ligation volume,

We calculated a j/i ratio of 3.84. Therefore, with the above assumptions, circularisation of DNA fragments is preferred over inter-molecular ligation during the inversion reaction.

  • In some instances when DNA concentrations are low (e.g. extracts from laser-microdissected tissue samples), we do not do the serial dilution step; instead we add the inverted DNA mixture directly to 1 mL of Amplitaq Gold PCR mix, vortex briefly and distribute 10 μL of PCR mix to each well, after which we proceed straight to the PCR step.
  • Serial dilutions should be mixed by slowly pipetting up and down 10 times using a 1.0 mL pipette set at ~100 μL. This will limit bubbles and cross-contamination of PCR products.
  • Heat the 96-pin replicator until the pins are red hot. Note that the heated 96-pin replicator will cause convection air currents, so cool for 5-10 min at RT before transferring the templates from the first PCR tray to the second. The replicator will transfer only ~1 μL. Some carbon flecks may also be transferred, but are sterile and should not affect the PCR.