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Method Article
David Hooker
Centre for Biomedical Research, Burnet Institute, 85 Commercial Rd, Melbourne, Victoria 3004, Australia.
Corresponding Author
License:
This work is licensed under a CC BY-NC 3.0 License. Read Full License
The finely orchestrated cell death process of apoptosis is fundamental to vertebrate and invertebrate health and growth, while its dysregulation contributes to infectious disease pathology and malignancy. Additionally, it is vital to evaluate the effects on apoptosis of drug candidates for cancer therapy. In view of the important role apoptosis plays in health, disease, drug design, therapy and its conservation across species, it is therefore highly beneficial to be able to measure apoptosis. In this article we describe ApoqPCR, a protocol to measure apoptosis by absolute quantitation of an apoptotic hallmark: internucleosomally fragmented (‘apoptotic’) genomic DNA. Completely apoptotic DNA is generated, diluted to cover a 1000-fold biologically relevant range of concentrations, amplified by ligation-mediated qPCR and threshold cycles measured. This forms an apoptotic DNA standard curve. A cell number standard curve is also constructed. Referencing test samples (with unknown amounts of apoptotic DNA) against each standard curve achieves an absolute value: the amount (picogram) of apoptotic DNA per cell number. ApoqPCR’s strengths include a broad linear 1000-fold dynamic range and an ability to measure apoptosis from sub-100 cell sample sizes. Additionally, ApoqPCR’s employment of an easily purified, stable, storable and re-useable starting material (nuclear genomic DNA) gives it value for archival/longitudinal studies and for apoptosis measurement from many vertebrate and invertebrate cell types. ApoqPCR requires genomic DNA of high and consistent quality; this precludes its use for degraded forensic samples. Because ApoqPCR measures from cell populations, the method is not suitable for monitoring single cell apoptosis and cannot in itself fractionate cell populations. In its present form and with standard curves established, 100 samples can be tested in triplicate by one operator in 5 days. The protocol is amenable to robotization which would increase through-put considerably.
Keywords
apoptosis, cell death, ApoqPCR, qPCR, PCR, absolute measurement, apoptotic DNA, internucleosomal DNA fragmentation, methods, aging, cancer, drug design
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The authors declare no competing financial interests.
This is a list of supplementary files associated with this preprint. Click to download.
- supplement0.pdf
Table 3 Reaction components and volumes for qLM-PCR reactions.
- supplement0.pdf
Table 4 Preparing a complex of Taq DNA polymerase and anti-Taq antibody.
- supplement0.pdf
Table 5 Cycling conditions for qLM-PCR.
- supplement0.pdf
Table 6 Construction of dilutions for the Cell Number standard curve.
- supplement0.pdf
Table 8 Cycling conditions for Cell Number qPCR.
- supplement0.pdf
Table 7 Reaction components and volumes for Cell Number qPCR.
- supplement0.pdf
Table 9 Troubleshooting ApoqPCR: Potential problems, reasons and possible solutions.
- supplement0.pdf
Table 1 Construction of dilutions for the Apoptotic DNA standard curve.
- supplement0.pdf
Table 2 Reaction components and volumes for annealing / ligation reactions.
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Posted 30 Sep, 2013
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Associated Publications
A new way of measuring apoptosis by absolute quantitation of inter-nucleosomally fragmented genomic DNA
by D. J. Hooker, M. Mobarok, J. L. Anderson, +6
Nucleic Acids Research (13 June, 2013)
Method Article
David Hooker
Centre for Biomedical Research, Burnet Institute, 85 Commercial Rd, Melbourne, Victoria 3004, Australia.
Corresponding Author
Version History
CURRENT STATUS: Posted
Version 1
Posted 30 Sep, 2013
Community comments: 2
Metrics
Comments: 0
HTML Views: ...
License:
This work is licensed under a CC BY-NC 3.0 License. Read Full License
The finely orchestrated cell death process of apoptosis is fundamental to vertebrate and invertebrate health and growth, while its dysregulation contributes to infectious disease pathology and malignancy. Additionally, it is vital to evaluate the effects on apoptosis of drug candidates for cancer therapy. In view of the important role apoptosis plays in health, disease, drug design, therapy and its conservation across species, it is therefore highly beneficial to be able to measure apoptosis. In this article we describe ApoqPCR, a protocol to measure apoptosis by absolute quantitation of an apoptotic hallmark: internucleosomally fragmented (‘apoptotic’) genomic DNA. Completely apoptotic DNA is generated, diluted to cover a 1000-fold biologically relevant range of concentrations, amplified by ligation-mediated qPCR and threshold cycles measured. This forms an apoptotic DNA standard curve. A cell number standard curve is also constructed. Referencing test samples (with unknown amounts of apoptotic DNA) against each standard curve achieves an absolute value: the amount (picogram) of apoptotic DNA per cell number. ApoqPCR’s strengths include a broad linear 1000-fold dynamic range and an ability to measure apoptosis from sub-100 cell sample sizes. Additionally, ApoqPCR’s employment of an easily purified, stable, storable and re-useable starting material (nuclear genomic DNA) gives it value for archival/longitudinal studies and for apoptosis measurement from many vertebrate and invertebrate cell types. ApoqPCR requires genomic DNA of high and consistent quality; this precludes its use for degraded forensic samples. Because ApoqPCR measures from cell populations, the method is not suitable for monitoring single cell apoptosis and cannot in itself fractionate cell populations. In its present form and with standard curves established, 100 samples can be tested in triplicate by one operator in 5 days. The protocol is amenable to robotization which would increase through-put considerably.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Loading...
Associated Publications
A new way of measuring apoptosis by absolute quantitation of inter-nucleosomally fragmented genomic DNA
by D. J. Hooker, M. Mobarok, J. L. Anderson, +6
Nucleic Acids Research (13 June, 2013)