Abstract
Whole-genome profiling using DNA arrays has led to tremendous advances in our understanding of cell biology. It has had similar success when applied to large viral genomes, such as the herpesviruses. Unfortunately, most DNA arrays still require specialized and expensive resources and, generally, large amounts of input RNA. An alternative approach is to query entire viral genomes using real-time quantitative PCR. We have designed such PCR-based arrays for every open reading frame of human herpesvirus 8 and describe here the general design criteria, validation procedures, and detailed application to quantify viral mRNAs. This should provide a useful resource either for whole-genome arrays or just to measure transcription of any one particular mRNA of interest. Because these arrays are RT-PCR-based, they are inherently more sensitive and robust than current hybridization-based approaches and are ideally suited to query viral gene expression in models of pathogenesis.
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References
Mullis, K. B. and Faloona, F. A. (1987) Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. Methods Enzymol. 155, 335–350.
Lomeli, H., Tygai, S., Pritchard, C. G., Lizardi, P. M., and Kramer, F. R. (1989) Quantitative assays based on the use of replicable hybridization probes. Clin. Chem. 35, 1826–1831.
Heid, C. A., Stevens, J., Livak, K. J., and Williams, P. M. (1996) Real time quantitative PCR. Genome Res. 6, 986–994.
Mackay, I. M., Arden, K. E., and Nitsche, A. (2002) Real-time PCR in virology. Nucleic Acids Res. 30, 1292–1305.
Fakhari, F. D. and Dittmer, D. P. (2002) Charting latency transcripts in Kaposi’s sarcoma-associated herpesvirus by whole-genome real-time quantitative PCR. J. Virol. 76, 6213–6223.
Dittmer, D. P. (2003) Transcription of Kaposi’s sarcoma-associated herpesvirus in Kaposi’s sarcoma lesions. Cancer Res. 63, 2010–2015.
Rozen, S. Primer3 Software Distribution. Retrieved on May 18, 2004. http://frodo.wi.mit.edu/primer3/primer_code.html. Boston, MA.
Santos, R. A., Hatfield, C. C., Cole, N. L., et al. (2000) Varicella-zoster virus gE escape mutant VZV-MSP exhibits an accelerated cell-to-cell spread phenotype in both infected cell cultures and SCID-hu mice [In Process Citation]. Virology 275, 306–317.
Lin, S. E, Robinson, D. R., Oh, J., et al. (2002) Identification of the bZIP and Rta homologues in the genome of rhesus monkey rhadinovirus. Virology 298, 181–188.
Rice, P., Longden, I., and Bleasby, A. (2000) EMBOSS: the European Molecular Biology Open Software Suite. Trends Genet 16, 276–277.
Bentley, D. (2002) The mRNA assembly line: transcription and processing machines in the same factory. Curr. Opin. Cell Biol. 14, 336–342.
Dittmer, D. P. (2003) Transcription profile of Kaposi’s sarcoma-associated herpesvirus in primary Kaposi’s sarcoma lesions as determined by real-time PCR arrays. Cancer Res. 63, 2010–2015.
Cantor, C. R. and Schimmel, P. R. (1980) Biophysical Chemistry: The Behavior of Biological Macromolecules. Freeman, New York.
Dittmer, D., Stoddart, C., Renne, R., et al. (1999) Experimental transmission of Kaposi’s sarcoma-associated herpesvirus (KSHV/HHV-8) to SCID-hu Thy/Liv mice. J. Exp. Med. 190, 1857–1868.
Glantz, S. and Slinker, B. (2000) Primer of Applied Regression & Analysis of Variance. McGraw-Hill, New York.
Pfaffl, M. W. (2001) A new mathematical model for relative quantification in realtime RT-PCR. Nucleic Acids Res. 29, E45–55.
Pfaffl, M. W., Horgan, G. W., and Dempfle, L. (2002) Relative expression software tool (REST) for group-wise comparison and statistical analysis of relative expression results in real-time PCR. Nucleic Acids Res. 30, e36.
Vandesompele, J., De Preter, K., Pattyn, F., et al. (2002) Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol. 3, RESEARCH0034.
Lossos, I. S., Czerwinski, D. K., Wechser, M. A., and Levy, R. (2003) Optimization of quantitative real-time RT-PCR parameters for the study of lymphoid malignancies. Leukemia 17, 789–795.
Eisen, M. B., Spellman, P. T., Brown, P. O., and Botstein, D. (1998) Cluster analysis and display of genome-wide expression patterns. Proc. Natl. Acad. Sci. USA 95, 14863–14868.
Bowtell, D. and Sambrook, J. (2003) DNA Microarrays: A Molecular Cloning Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, p. 712.
Thomas, D. and Hunt, A. (2001) Programming Ruby. Addison-Wesley, Boston.
Hwang, S., Lee, D., Gwack, Y., Min, H., Choe, J. (2003) Kaposi’s sarcoma-associated herpesvirus K8 protein interacts with hSNF5. J. Gen. Virol. 84, 665–676.
Lallemand, F., Desire, N., Rozen Daum, W., et al. (2000) Quantitative analysis of human herpesvirus 8 viral load using a real-time PCR assay. J. Clin. Microbiol. 38, 1404–1408.
White, I. E. and Campbell, T. B. (2000) Quantitation of cell-free and cell-associated Kaposi’s sarcoma-associated herpesvirus DNA by real-time PCR. J. Clin. Microbiol. 38, 1992–1995.
Schweiger, B., Zadow, I., Heckler, R., et al. (2000) Application of a fluorogenic PCR assay for typing and subtyping of influenza viruses in respiratory samples. J. Clin. Microbiol. 38, 1552–1558.
Kutyavin, I. V., Afonia, I. A., Mills, A., et al. (2000) 3′-Minor groove binder-DNA probes increase sequence specificity at PCR extension temperatures. Nucleic Acids Res. 28,. 655–661.
Afonina, I., Zivarts, M., Kutyavin, I. V., et al. (1997) Efficient priming of PCR with short oligonucleotides conjugated to a minor groove binder. Nucleic Acids Res. 25, 2657–2660.
Buhr, C. A., Wagner, R. W., Grant, D., and Froeheen, B. C. (1996) Oligodeoxynucleotides containing C-7 propyne analogs of 7-deaza-2′-deoxyguanosine and 7-deaza-2′-deoxyadenosine. Nucleic Acids Res. 24, 2974–2980.
Wagner, R. W., Matteucci, M. D., Lewis, J. G., et al. (1993) Antisense gene inhibition by oligonucleotides containing C-5 propyne pyrimidines. Science 260, 1510–1513.
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Papin, J., Vahrson, W., Hines-Boykin, R., Dittmer, D.P. (2005). Real-Time Quantitative PCR Analysis of Viral Transcription. In: Lieberman, P.M. (eds) DNA Viruses. Methods in Molecular Biology, vol 292. Humana Press. https://doi.org/10.1385/1-59259-848-X:449
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DOI: https://doi.org/10.1385/1-59259-848-X:449
Publisher Name: Humana Press
Print ISBN: 978-1-58829-353-4
Online ISBN: 978-1-59259-848-9
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