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Department of Pathology, School of Medicine, 50 North Medical Dr., University of Utah, Salt Lake City, UT 84132.
2
ARUP Institute for Clinical and Experimental Pathology,
500 Chipeta Way, Salt Lake City, UT 84108.
3
Department of Pathology and Feist-Weiller Cancer Center,
Louisiana State University Health Sciences Center, 1501 Kings Hwy.,
Shreveport, LA 71130.
4
Idaho Technology, 390 Wakara Way, Salt Lake City, UT
84108.
aAddress correspondence to this author at: ARUP Laboratories, 500 Chipeta Way, Salt Lake City, UT 84108. Fax 801-584-5207; e-mail lyone{at}aruplab.com.
Background: Molecular detection methods for HER2/neu gene amplification include fluorescence in situ hybridization (FISH) and competitive PCR. We designed a quantitative PCR system utilizing fluorescent hybridization probes and a competitor that differed from the HER2/neu sequence by a single base change.
Methods: Increasing twofold concentrations of competitor were coamplified with DNA from cell lines with various HER2/neu copy numbers at the HER2/neu locus. Competitor DNA was distinguished from the HER2/neu sequence by a fluorescent hybridization probe and melting curve analysis on a fluorescence-monitoring thermal cycler. The percentages of competitor to target peak areas on derivative fluorescence vs temperature curves were used to calculate copy number.
Results: Real-time monitoring of the PCR reaction showed comparable relative areas throughout the log phase and during the PCR plateau, indicating that only end-point detection is necessary. The dynamic range was over two logs (2000–250 000 competitor copies) with CVs <20%. Three cell lines (MRC-5, T-47D, and SK-BR-3) were determined to have gene doses of 1, 3, and 11, respectively. Gene amplification was detected in 3 of 13 tumor samples and was correlated with conventional real-time PCR and FISH analysis.
Conclusion: Use of relative peak areas allows gene copy numbers to be quantified against an internal competitive control in <1 h.
The following articles in journals at HighWire Press have cited this article:
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  E. Lyon and C. T. Wittwer LightCycler Technology in Molecular Diagnostics J. Mol. Diagn., March 1, 2009; 11(2): 93 - 101. [Abstract] [Full Text] [PDF]
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 C. D'Addario, Y. Ming, S.-O. Ogren, and L. Terenius The role of acetaldehyde in mediating effects of alcohol on expression of endogenous opioid system genes in a neuroblastoma cell line FASEB J, March 1, 2008; 22(3): 662 - 670. [Abstract] [Full Text] [PDF]
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 S. Rauser, R. Weis, H. Braselmann, M. Feith, H. J. Stein, R. Langer, P. Hutzler, M. Hausmann, S. Lassmann, J. R. Siewert, et al. Significance of HER2 Low-Level Copy Gain in Barrett's Cancer: Implications for Fluorescence In situ Hybridization Testing in Tissues Clin. Cancer Res., September 1, 2007; 13(17): 5115 - 5123. [Abstract] [Full Text] [PDF]
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 J. M. Flowers and R. S. Burton Ribosomal RNA Gene Silencing in Interpopulation Hybrids of Tigriopus californicus: Nucleolar Dominance in the Absence of Intergenic Spacer Subrepeats Genetics, July 1, 2006; 173(3): 1479 - 1486. [Abstract] [Full Text] [PDF]
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K. A. Warner, E. L. Crawford, A. Zaher, R. J. Coombs, H. Elsamaloty, S. L. Roshong-Denk, I. Sharief, G. V. Amurao, Y. Yoon, A. Y. Al-Astal, et al. The c-myc x E2F-1/p21 Interactive Gene Expression Index Augments Cytomorphologic Diagnosis of Lung Cancer in Fine-Needle Aspirate Specimens J. Mol. Diagn., August 1, 2003; 5(3): 176 - 183. [Abstract] [Full Text] [PDF]
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A. Millson, A. Suli, L. Hartung, S. Kunitake, A. Bennett, M. C. L. Nordberg, W. Hanna, C. T. Wittwer, A. Seth, and E. Lyon Comparison of Two Quantitative Polymerase Chain Reaction Methods for Detecting HER2/neu Amplification J. Mol. Diagn., August 1, 2003; 5(3): 184 - 190. [Abstract] [Full Text] [PDF]
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 G. Pont-Kingdon and E. Lyon Rapid Detection of Aneuploidy (Trisomy 21) by Allele Quantification Combined with Melting Curves Analysis of Single-Nucleotide Polymorphism Loci Clin. Chem., July 1, 2003; 49(7): 1087 - 1094. [Abstract] [Full Text] [PDF]
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M. Konigshoff, J. Wilhelm, R. M. Bohle, A. Pingoud, and M. Hahn HER-2/neu Gene Copy Number Quantified by Real-Time PCR: Comparison of Gene Amplification, Heterozygosity, and Immunohistochemical Status in Breast Cancer Tissue Clin. Chem., February 1, 2003; 49(2): 219 - 229. [Abstract] [Full Text] [PDF]
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 M. Stocher and J. Berg Normalized Quantification of Human Cytomegalovirus DNA by Competitive Real-Time PCR on the LightCycler Instrument J. Clin. Microbiol., December 1, 2002; 40(12): 4547 - 4553. [Abstract] [Full Text] [PDF]
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N. Wedemeyer, T. Potter, S. Wetzlich, and W. Gohde Flow Cytometric Quantification of Competitive Reverse Transcription-PCR Products Clin. Chem., September 1, 2002; 48(9): 1398 - 1405. [Abstract] [Full Text] [PDF]
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S. M. Williams, C. A. Meadows, and E. Lyon Automated DNA Extraction for Real-Time PCR Clin. Chem., September 1, 2002; 48(9): 1629 - 1630. [Full Text] [PDF]
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