1 | Purify test sample genomic DNA
Critical Some cell types and tissues, for example adipose tissue, may require specialised protocols for gDNA purification that are not outlined here. We recommend that a standard purification procedure be applied to all samples to be tested after any specialised purification protocols have been completed, to minimise irregularities between samples. This standard purification procedure can be column-based, should remove protein and RNA and purify DNA within the range 150 bp to 30,000 bp. Quantitative recovery is not necessary.
1.1) Follow manufacturer recommendations including RNA’ase treatment. Using Qiagen QIAamp DNA Mini Kit columns or equivalent, purify per column up to 200 µl blood, plasma, serum, buffy coat, up to 5 x 106 cultured cells, or up to 25 mg tissue (for eg, liver, brain, lung, heart, kidney, or up to 10 mg spleen). Elute in 200 to 250 µl 10 mM Tris-HCl pH 8.5, 0.5 mM EDTA (Qiagen Buffer AE or TE, PCR grade). Expected yields are 20 to 250 ng/µl depending on the blood cell count, culture conditions or tissue type.
1.2) Measure the DNA concentration using a spectrophotometer. Adjust to about 5 ng/µl in TE for ligation-mediated qPCR as this concentration permits a wide range of (unknown) apoptotic DNA levels to be measured when referenced to the standard curve.
1.3) Store gDNA samples at -80°C.
Critical If repeated access to samples is anticipated aliquot the gDNA into 30 µl volumes to avoid repeated freeze-thawing. Freeze-thawing the gDNA more than 3 times can affect results, possibly due to damage to very low-abundance species of apoptotic DNA. Do not store gDNA in water to avoid acid hydrolysis. Archived tissue is suitable for ApoqPCR as long as storage conditions are correct. In this case prompt storage at -80°C and prompt gDNA purification post-thawing is the minimum required.
2 | Generate completely apoptotic DNA from a standardized source for the construction of an Apoptotic DNA standard curve
2.1) Culture Jurkat cells to log phase (with acidity indicator not permitted to turn yellow) in 40 ml RF-10 in a T75 flask at 37°C / 5% CO2 within a humidified cell culture incubator.
2.2) Pellet cells by centrifugation at room temperature (RT), 450 g for 10 min. Resuspend in fresh, pre-warmed RF-10 and count accurately (haemocytometer) then adjust with the same medium to give 1.0 x 106 cells /ml. Distribute 20 ml to a pre-warmed T75 flask for incubation with staurosporine and, if desired, the same volume to another T75 flask to serve as a negative control (no staurosporine).
Caution Staurosporine can cause external and internal irritation. Wear personal protective equipment and work in a biological containment hood.
2.3) To the pre-warmed cell suspensions (37°C), add 160 µl of 1000 µM staurosporine to give 8.0 µM, gently mix thoroughly into solution and immediately incubate flasks at 37°C for 5.0 hr. Every hour gently agitate the flasks by hand, avoiding cooling of the cultures.
2.4) For each 20 ml culture at 5.0 hr, re-suspend settled cells by gentle agitation, collect as multiples of 1.3 ml into eppendorf tubes, pellet at 450 g for 6 min at RT, resuspend each pellet in 200 µl phosphate buffered saline (PBS).
Pause point These suspensions can be quick frozen at -80°C to store indefinitely if needed. Otherwise proceed immediately to gDNA purification.
2.5) Purify the apoptotic DNA from a 200 µl cell suspension in PBS (equivalent to 1.3 ml cultured cells) with one Qiagen QIAamp DNA mini column following manufacturer’s recommendations including RNA’ase treatment (as for test gDNA), elute in 200 µl TE and quantify spectro-photometrically. Expected yields are in the range ~30 to 50 ng/µl.
Pause point Either store at -80°C and thaw once before proceeding, or proceed to serial dilution (see below).
3 | Verify conversion of genomic DNA to completely apoptotic DNA
3.1) Electrophorese 0.5 or 1 µg fractions of completely apoptotic DNA on a 1.5% agarose 1x Tris-Acetate-EDTA (TAE) gel. Stain for 50 min with ethidium bromide at 15 µg/ml then destain for 30 min in double-distilled water, or alternatively incorporate SYBR Safe at 1x in the gel.
Critical The quality of information obtained from trace intensity profiles depends on agarose gel quality. Pour gels that are free from particulate matter and load samples precisely into wells to acquire a dependable electrophoretic image.
3.2) For post-electrophoretic trace intensity visualisation and quantification, digitally capture images over whole gels and use exposure settings that avoid pixel saturation. For the BioRad Gel-Doc Imager we set the sensitivity at 10.00, normalisation off. Frame lanes, detect bands manually and subtract lane background by the rolling circle method. For background subtraction, select the rolling circle radii to preserve complete trace profile area for all bands. Trace quantity of a band is the measured area under its trace profile curve in units of intensity x mm2. With this software, only non-Gaussian trace quantification is applicable due to the shape and spacing of bands. "Figure 3":http://www.nature.com/protocolexchange/system/uploads/2688/original/Figure_3.jpg?1379817776 shows typical electrophoretic profiles. With completely apoptotic DNA prepared this way we observe by trace profiles that no genomic DNA remains unconverted to apoptotic DNA. DNA needs to be essentially completely apoptotic so that spectrophotometric quantitation is accurate.
4 | (Optional) Additional steps to verify the conversion of genomic DNA to completely apoptotic DNA
4.1) We have used a modified TUNEL procedure and subsequent measurement of the TUNEL-positive cell percentage by flow cytometry to further confirm the extent of conversion to apoptotic DNA.
4.2) From the 20 ml cultures above and at the 5 hr incubation point, collect 10 ml (107) cells into eppendorf tubes. Pellet 450 g for 5 min, RT.
4.3) Wash 2x at RT in PBS, resuspend total cells to 2 x 107 cells /ml in 500 µl PBS and transfer 95 µl cells to triplicate wells of a V-bottom plate.
Pause Point Cells can be kept on ice in the 500 µl PBS for ≤ 1 hr before transferral to V-bottom plate.
4.4) Pellet plated cells at 450 g (1200 rpm) in a Beckman GS6R centrifuge or equivalent for 10 min at RT and aspirate supernatant carefully to avoid disrupting pellets.
4.5) Add 100 µl per well Cytofix / Cytoperm solution. Resuspend cells by pipetting up and down gently 5 times then incubate 20 min at 4°C on nutator mixer.
4.6) Wash 2x with 200 µl / well of 1x Perm/Wash buffer by pipetting up and down gently 5 times and centrifuge as above.
4.7) During washes prepare TUNEL reaction mixture according to instructions (Roche kit). Keep on ice until added to wells. Each set equals 3 test triplicate wells, 1 autofluorescence well (cells, no label, no enzyme), and 1 negative control well (cells, +label, no enzyme), ie 5 wells per set.
4.8) To test triplicate wells add 50 µl per well TUNEL reaction mixture (label + enzyme). To the autofluorescence well add 200 µl 1x Perm/Wash buffer. To the negative control add 50 µl label solution. Resuspend gently.
4.9) Add lid and incubate 60 min at 37°C in a dark humidified atmosphere.
4.10) Pellet cells as above.
4.11) Wash 2x with 200 µl/well 1x Perm/Wash buffer as before then resuspend cells in 250 µl 1% paraformaldehyde in PBS.
Pause Point Samples can be stored in the dark at 4°C overnight for processing the next day.
4.12) Process cells by flow cytometry ideally within the hour. Perform flow cytometry at excitation 488nm, detection 530nm, gate to exclude sub-cellular debris and select the total cell population. Sort at least 10,000 events at each measurement.
5 | Construct serial dilutions of completely apoptotic DNA for the Apoptotic DNA standard curve
5.1) Adjust 100% apoptotic DNA to 23.15 ng/µl with TE. Prepare 4-fold serial dilutions from 9.26 ng/µl (40%) to 9.03 pg/µl covering a 1000-fold range (6 serial dilutions). ‘40%’ apoptotic DNA is chosen as a starting dilution because, from experience, samples generated in vitro and especially clinical samples rarely exceed this upper level. See "Table 1":http://www.nature.com/protocolexchange/system/uploads/2693/original/Table_1.pdf?1379820818 for a suggested dilution scheme.
5.2) Mix each dilution thoroughly. Dispense single-use 20 to 30 µl aliquots for each of these dilutions into air-tight tubes. Ensure all liquid is at base of tube then store at -80°C in a non-cyclic defrost freezer prior to use in annealing/ligation reactions.
Critical Single use aliquots are necessary to avoid freeze-thaws which would change threshold values. The use of a non-cyclic defrost freezer prevents dehydration.
6 | Measure the unknown amount of apoptotic DNA in a test sample of genomic DNA by ligation-mediated qPCR (qLM-PCR).
6.1) Prepare and perform annealing/ligation reactions using the 6 apoptotic DNA serial dilutions (for the standard curve) and test sample gDNA and no template control (PCR-grade water). Annealing/ligation reactions for the standard curve are constructed such that the amount of apoptotic DNA in each 24 µl reaction ranges from 160.0 ng to 156.3 pg.
6.1.1) Thaw, mix and pulse spin the apoptotic DNA serial dilutions and test sample gDNA.
6.1.2) Assemble reactions for the 6 apoptotic DNA serial dilutions and test sample gDNA and no template control in thin-walled 200µl capacity PCR tubes. See "Table 2":http://www.nature.com/protocolexchange/system/uploads/2694/original/Table_2.pdf?1379820913.
Critical Calibrated pipettes that accurately deliver sub-20 µl and sub-2 µl volumes are essential. Perform all liquid handling under PCR conditions. Due to the presence of polyethylene glycol, reactions must be mixed thoroughly to homogeneity.
Critical Linker synthesis by different manufacturers can dramatically influence the threshold cycle and replicate reproducibility by changing background fluorescence during qPCR. This may be due to extraneous solute material carried over during synthesis. The manufacturer used here is given in Reagents and Equipment.
6.1.3) Carefully place tubes containing assembled reaction components in a PCR thermal cycler avoiding any droplets on tube sides.
6.1.4) Anneal linkers under the following programmed conditions (or similar):
55°C for 10 min, 50°C to 15°C in 5°C/8 min increments, 10°C for 20 min (see 6.1.5 below), 16°C for 16 hr, 2°C to 4°C on hold.
Critical Machine-control the temperature gradient for annealing runs to avoid inter-run variability. A ‘Pause’ function in the cycler software is necessary.
6.1.5) For ligation of annealed linkers to apoptotic target DNA, halfway through the 10°C step (ie. at the 10 min point), pause the program and to each PCR tube, add 2.4 U of 1 U /µl T4 DNA ligase, mix, close the tube, then when the series of tubes is completed un-pause the program to complete the 10°C incubation for the remaining 10 min, ramp to 16°C for 16 hr for ligation (overnight), then 2 to 4°C on hold.
Critical The addition of T4 DNA ligase occurs to reaction tubes while they reside in the thermal cycler heating block. Open each tube in turn and manually add ligase. Mixing is critical especially considering the presence of viscous polyethylene glycol. Add, expel and mix ligase with the pipette tip, adopting the same manual procedure for each tube to aid reproducibility. We have found that ‘extended length’ filter tips on a Gilson P10 hinder complete expulsion, while standard length P10 tips assist expulsion.
6.1.6) After the 16 hr ligation, dilute all 24 µl annealed/ligated reactions (now 26.4 µl due to ligase addition) to 80 µl with 53.6 µl TE. Mix thoroughly. Note that 7.50 µl from these reactions will be used 3 times for 3 replicates in the qLM-PCR reactions.
Pause point The completed and diluted annealed/ligated reactions for test samples are normally used for one set of triplicate qLM-PCR runs. However, they can be frozen/thawed once at -20°C or -80°C for a repeat run on another day without significant change to threshold cycles.
6.2) Prepare and perform qLM-PCR.
6.2.1) Prepare qLM-PCR master mix as in "Table 3":http://www.nature.com/protocolexchange/system/uploads/2695/original/Table_3.pdf?1379820960 and "Table 4":http://www.nature.com/protocolexchange/system/uploads/2696/original/Table_4.pdf?1379821032.
6.2.2) Assemble qLM-PCR reactions for test sample, standard curve dilution or no-template control. Use 3 x 7.5 µl of the diluted annealed/ligated reactions in triplicate 25 µl qLM-PCR reactions. See "Table 3":http://www.nature.com/protocolexchange/system/uploads/2695/original/Table_3.pdf?1379820960. Ninety-six well qPCR plates are used, permitting the 6 standard curve dilutions, one no template control and a maximum of 25 test samples all run in triplicate. For the six 4-fold diluted standards, qLM-PCR reactions contain 15,000 pg down to 14.6 pg completely apoptotic DNA.
To do this, first add 17.5 µl of the master mix into each well of the qPCR plate without employing the pipette’s second stage expulsion step (this eliminates unwanted bubbles while still being reproducible, and is faster). Then add 7.5 µl of the appropriate diluted
annealed/ligated reaction per well. When adding diluted annealed/ligated reactions to wells, for each well pipette up and down a consistent number of times, finishing with a second stage expulsion.
6.3) Perform qLM-PCR in a qPCR thermal cycler.
6.3.1) Before cycling, spin the qPCR plate with assembled reactions at about 200 g for 5 sec to ensure all reaction contents are at the base of the wells.
6.3.2) Cycling conditions are given in "Table 5":http://www.nature.com/protocolexchange/system/uploads/2697/original/Table_5.pdf?1379821703. Program the thermal cycler to read fluorescence at the end of the 72°C / 3 min annealing-extension step.
7 | Purify genomic DNA from a standardised, diploid and accurately counted cell source for the construction of a Cell Number standard curve
7.1) Using approximately 100 ml blood from a healthy donor, isolate peripheral blood mononuclear cells (PBMC) by Ficoll density gradient centrifugation. To do this, onto 6 x 10 ml Ficoll in 6 sterile 50 ml tubes, layer equal volumes of blood.
Caution Blood must be handled, contained and discarded as a biohazard. Wear personal protective equipment and perform all work under PC2 conditions in a bio-containment cabinet.
Critical Use primary cells with diploid genomes. Cell lines are frequently heteroploid and therefore cannot be used.
7.2) Separate blood components by centrifugation at RT, 750 g for 30 min without brake.
7.3) Transfer the white PBMC layers to 2 new tubes. Wash 4 times at RT with 30 ml PBS washes per tube to remove platelets, centrifuging at 250 g for 15 min at RT with moderate brake. After final wash, remove supernatant and gently resuspend total cells in 1.0 ml PBS at RT.
Critical Several washes are needed to permit accurate cell counts.
7.4) Count cells (if with haemocytometer, mean of 4 counts), pellet cells in eppendorf tube, remove supernatant and resuspend cells to 6 x 107 /ml in freshly prepared lysis buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA, PCR grade water; plus final concentrations of 0.002% Triton X-100, 0.002% sodium dodecyl sulphate (SDS) and 0.8 mg/ml proteinase K added just before use).
7.5) Lyse cells at 56°C for 1 hr with occasional gentle agitation, then inactivate proteinase K at 95°C for 15 min. Cool the lysate to RT.
Critical Proteinase K must be totally inactivated to avoid interference in downstream reactions.
7.6) Create six 5-fold serial dilutions from 50,000 cell-equivalents per 1.62 µl to 16 cell-equivalents per 1.62 µl using TE as diluent. A suggested dilution series is given in "Table 6":http://www.nature.com/protocolexchange/system/uploads/2698/original/Table_6.pdf?1379821763.
7.7) Divide into 20 to 30 µl single-use aliquots and store immediately in a non-cyclic defrost freezer at -80°C prior to use.
Critical Storage of single-use aliquots at -80°C is necessary to avoid degradation of target DNA.
8 | Measure the unknown cell number equivalence in a test sample by qPCR, from the same amount of genomic DNA used in the apoptotic DNA reactions
8.1) Prepare and perform Cell Number qPCR.
8.1.1) Prepare Cell Number qPCR master mix as in "Table 7":http://www.nature.com/protocolexchange/system/uploads/2699/original/Table_7.pdf?1379821812.
8.1.2) Assemble Cell Number qPCR reactions for test sample, standard curve dilution or no-template control. Ninety-six well qPCR plates are used, permitting the 6 standard curve dilutions, one no template control and a maximum of 25 test samples all run in triplicate. Pipette 23.38 µl of the master mix into each well without employing the pipette’s second stage expulsion step (this eliminates unwanted bubbles while still being reproducible, and is faster).
8.1.3) Then add 1.62 µl of each standard / test gDNA / no template control per well, as in "Table 7":http://www.nature.com/protocolexchange/system/uploads/2699/original/Table_7.pdf?1379821812, and mix by pipetting up and down 5 times, finishing with a second-stage expulsion.
Critical The volume of 1.62 µl test sample gDNA is critical because it means that the same amount of test sample gDNA is added to both qLM-PCR and Cell Number qPCR reactions. Use high quality, calibrated pipettes under PCR conditions.
8.1.4) Before qPCR cycling, spin the capped qPCR plate at about 200 g for 5 sec to ensure all reaction contents are at the base of the wells.
8.1.5) Perform Cell Number qPCR with cycling conditions as shown in "Table 8":http://www.nature.com/protocolexchange/system/uploads/2700/original/Table_8.pdf?1379821867.