Clinical Chemistry
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Clinical Chemistry 43: 752-758, 1997;
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (59)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Gelmini, S.
Right arrow Articles by Pazzagli, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Gelmini, S.
Right arrow Articles by Pazzagli, M.
Related Collections
Right arrow Molecular Diagnostics and Genetics
(Clinical Chemistry. 1997;43:752-758.)
© 1997 American Association for Clinical Chemistry, Inc.

Articles

Quantitative polymerase chain reaction-based homogeneous assay with fluorogenic probes to measure c-erbB-2 oncogene amplification

Stefania Gelminia, Claudio Orlando, Roberta Sestini, Giovanna Vona, Pamela Pinzani, Luca Ruocco1 and Mario Pazzagli

1 Perkin-Elmer Italy, Monza, Milan, Italy.
a Author for correspondence. Fax +39.55.4377290.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We describe a PCR-based assay for determining c-erbB-2 oncogene amplification in breast cancer in which we use the TaqManTM system. Two fluorogenic probes anneal to the target between primers for c-erbB-2 and ß-globin genes and contain both a reporter dye (6-carboxy-fluorescein) and a quencher dye (6-carboxy-tetramethyl-rhodamine). During the extension phase of the PCR cycle, the 5'->3' exonuclease activity of Taq polymerase cleaves the hybridized fluorogenic probe, resulting in an increase of fluorescence emission of the reporter dye that is quantitative for the amount of PCR product and, under appropriate conditions, for the amount of template. Assay performance showed adequate precision and a lower detection limit and good correlation with the results obtained in the same samples by a competitive PCR assay (n = 25, r = 0.94, P <0.01). This homogeneous assay is time-saving, avoids usually cumbersome postamplification procedures (that can be additional sources of inaccuracy and contamination), and seems suitable for determination of c-erbB-2 oncogene amplification in tumor specimens.


Key Words: indexing terms: breast cancer • genetic alterations • oligonucleotide probes • fluorescence assays


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Several processes for quantitative PCR have been proposed. These approaches have been addressed by either assessments of procedures for accurate measurement of PCR products (1)(2)(3)(4)(5) or strategies involving coamplification of an external reference gene (differential PCR) (6)(7) or an internal standard (competitive PCR) (8)(9)(10). Independently from the various strategies adopted, however, measurements of a nucleic acid target (DNA or mRNA) by PCR require time-consuming postamplification steps that can, to some extent, introduce additional uncontrolled variables and risks of carryover contamination. Thus, any evolution of PCR techniques that allows direct detection of specific amplified targets without any post-PCR processing is relevant.

Recently, detection of specific PCR products with fluorogenic probes has been proposed (11)(12)(13)(14). The probes are designed to hybridize within the target sequence and to generate a signal that accumulates during PCR cycling in proportion to the accumulation of amplification products. The end-point measurement of fluorescence in each sample thus provides a homogeneous signal that is specifically associated with the amplified target and quantitatively related to the amount of PCR products, without requiring further post-PCR procedures.

To verify the applicability of this approach to quantitative PCR, we developed an assay for measuring c-erbB-2 amplification in DNA from human breast tumors. Protooncogene amplification has been consistently observed in human tumors and, although no direct correlation between gene amplification and the pathogenic mechanisms of tumorigenesis has been clearly established, in several cases detection of oncogene amplification can serve as a genetic marker for prognosis. In particular, several authors have reported a direct correlation between c-erbB-2 amplification in breast carcinoma and clinical outcome (15)(16)(17)(18).

In this study, we used the instrumentation and the fluorogenic probes of the Perkin-Elmer Cetus (Norwalk, CT) LS-50B TaqManTM System (19). The results were compared with those of a previously assessed competitive PCR method for measuring c-erbB-2 oncogene amplification (8)(9).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
"taqman" principle and design of fluorogenic probes
The principle of the TaqMan LS-50B system is schematically summarized in Fig. 1 . Specific oligonucleotide probes are designed to anneal to the target between the two PCR primers (panel A, top). The probe contains 6-carboxy-fluorescein as the fluorescent reporter dye covalently linked to the 5' end. The quencher dye, 6-carboxy-tetramethyl-rhodamine, is usually covalently linked close to the 3' end. In addition, the probe contains a 3'-blocking phosphate group to prevent probe extension during PCR cycling (19). The closeness of the quencher to the reporter emitter means that the reporter fluorescence is suppressed, mainly by Förster-type energy transfer (20)(21) (Fig. 1B ). During PCR cycling, the probe specifically hybridizes to the corresponding template and then is cleaved via the 5'->3' exonuclease activity of Taq DNA polymerase (19) (Fig. 1A ). This cleavage results in an increase of fluorescence emission of the 6-carboxy-fluorescein reporter dye, without affecting the emission of quencher dye (Fig. 1B ). This sequence of events occurs in each PCR cycle, in the enzymatic reaction, and in the PCR product accumulation. Because the exonuclease activity of the Taq polymerase acts only if the fluorogenic probe is annealed to the target (but cannot hydrolyze the probe free in solution), the increase of fluorescence is proportional to the amount of the specific PCR product.



View larger version (18K):
[in this window]
[in a new window]
  		Figure 1. Schematic representation of fluorescence emission by TaqMan probe reaction.

During PCR annealing, the fluorogenic oligonucleotide specifically hybridizes to the corresponding template (A,top); the probe is then cleaved via the 5'->3' endonucleolytic activity of Taq polymerase, during PCR extension (A, bottom). The release of the reporter dye (R) from the 5' end and its consequent separation from the quencher dye (Q) generates a specific fluorescence emission at 518 nm (B, bottom), but no signal is emitted when the probe is intact (B, top). The probe is then displaced from the target, and the polymerization of the strand continues.

The sequence of the probes utilized in this report to detect ß-actin, ß-globin, and c-erbB-2 genes is reported in Table 1 . Probes were designed according to the manufacturer's guidelines (19). Briefly, the TaqMan probe should (a) be 20–40 bases long, to ensure good hybridization and specificity of binding; (b) have a GC content of 40–60% and avoid long runs of a single nucleotide; and (c) neither hybridize nor overlap with the forward and reverse primers. It is important to design a probe that forms a more stable hybrid than do the PCR primers; thus, the melting temperature (Tm) of the probe should be at least 5 °C higher than that of the PCR primers. Synthesis and purification of the fluorogenic probes was performed by Perkin-Elmer Cetus. The detection system for ß-actin gene, provided by the manufacturer as "reference detection reagents" (code 401846), was used for preliminary assessment of the PCR conditions.


View this table:
[in this window]
[in a new window]
  		Table 1. Primer and probe sequences of ß-actin, c-erbB-2, and ß-globin genes.

tumor samples and dna extraction
We used the proposed method to measure amplification of c-erbB-2 oncogene in 25 primary breast tumors (amplification range 1–22-fold), in which the presence of this genetic alteration had already been determined with a previously described competitive PCR procedure (8)(9), and in DNA specimens obtained from formalin-fixed, paraffin-embedded breast tumor. DNA was extracted from archived samples after deparafinization, as reported elsewhere (22), by a routine phenol–chloroform procedure (23).

Histopathological classification of the tumor samples was as follows: The tumor stage was pT1a in 2 cases, pT1b in 3, pT1c in 9, pT2 in 9, and pT4 in 2 cases; the nuclear grade was G3 in 14 cases, G2 in 9, and G1 in 2. Eighteen patients showed nodal involvement at the time of surgery.

pcr conditions
For PCR amplification, we utilized a Model 480 thermal cycler from Perkin-Elmer Cetus. The 10x PCR Buffer; the mononucleotides dATP, dCTP, dGTP, and dTTP; and the AmpliTaq DNA Polymerase were also from Perkin-Elmer. The PCR mix used for the TaqMan system was: 10 mmol/L Tris-HCl (pH 8.3), 50 mmol/L KCl, 1.5 mmol/L MgCl2 (3.5 mmol/L MgCl2 for the ß-actin gene), 0.2 mmol/L of each dNTP, 0.3 µmol/L of each primer, 0.2 µmol/L of each probe, and 1.25 U of AmpliTaq (Perkin-Elmer Cetus) in a 50-µL final volume. The PCR cycles for ß-globin and for c-erbB-2 were: 30 s at 94 °C, 30 s at 60 °C, and 30 s at 72 °C. For ß-actin amplification, the PCR conditions were: 30 s at 94 °C, 30 s at 54 °C, and 30 s at 72 °C. Primer sequences are reported in Table 1Up .

fluorescence measurement and calculations
The fluorescence was measured with a luminescence spectrometer (Model LS-50B; Perkin-Elmer) equipped with a plate reader (see Fig. 2 ). We adopted a standard configuration for use in the three assays: 488 nm excitation wavelength; 518 and 580 nm emission wavelengths for the reporter and quencher dyes, respectively; and a 2-s integration time. After PCR cycling, a maximum of 40 µL from each sample or blank was transferred to a microwell.



View larger version (118K):
[in this window]
[in a new window]
  		Figure 2. (Top) The luminescence spectrometer (Perkin-Elmer Cetus LS-50B) used for fluorescence measurement of the TaqMan-generating system; (bottom) the 96-well plate reader.

As previously described [19], the fluorescent signal results from changes in the fluorescence emission intensity of the reporter dye after the cleavage of the probe. Interfering fluorescence fluctuations are normalized by applying two calculations. In the first, the quencher dye is a passive internal standard; we divide the emission intensity of the reporter dye by the emission intensity of the quencher dye for each reaction to give a ratio defined as the RQ+ value. Nonspecific effects, e.g., concentration changes resulting from volume fluctuations, are normalized by this ratio. Any other fluctuation, besides that attributable to PCR-related nuclease digestion, is normalized by taking the RQ+ value for a sample tube that contains all components (including target DNA) and subtracting from this the value of the "no-template" control tube, i.e., containing all of the same components except template and defined as RQ-. This final {Delta}RQ value (i.e., RQ+ - RQ-) reliably indicates the magnitude of the signal generated by the given set of PCR conditions (19). Calculations and procedures for fluorescence signal acquisition are obtained with a personal computer connected on-line with the LS-50B luminescence spectrometer and equipped with a proprietary software specifically developed for the TaqMan technology.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
effect of pcr cycle number and dna quantity on taqman reaction: ß-actin calibration
The preliminary requirement of a PCR-based quantitative assay performed in the absence of an internal standard is to maintain the exponential phase of product accumulation, which is influenced by several known variables (number of cycles, DNA target concentration) and by certain unpredictable variables (mainly associated with the quality of the samples). In addition, in the method reported here, PCR product yield also depends on the efficiency of the cycling reaction of hybridization and on the cleavage of the fluorogenic probe. For these reasons, PCR conditions were previously assessed for the ß-actin gene (described as "reference detection reagents" in TaqMan principle and design of fluorogenic probes), with respect to the number of PCR cycles and the dose dependence of product accumulation. To verify the TaqMan PCR dependence on the number of PCR cycles, we amplified the ß-actin gene, starting with three quantities of normal target DNA (2.5, 10, and 40 ng) and using various numbers of PCR cycles. Evaluating the fluorescence emission (RQ+) associated with PCR product accumulation, we observed a general linear relationship between number of cycles and generated signal in the range 28–34 cycles for all three starter DNA quantities (Fig. 3 A). However, evaluation of the linear response of {Delta}RQ measured at 28, 31, and 34 cycles, as related to the amount of target DNA, showed a better dose-dependent response in the range between 28 and 31 cycles (Fig. 3B ). Consequently, we performed the subsequent experiments with a standard 30-cycle PCR amplification.



View larger version (18K):
[in this window]
[in a new window]
  		Figure 3. Dependency of TaqMan fluorescence emission as a function of DNA target quantities and PCR cycle number: (A) ß-actin gene amplification, expressed as RQ+ values, performed with increasing numbers of PCR cycles, starting from three quantities of normal DNA and a no-template sample; (B) linear {Delta}RQ response at 28, 31, and 34 PCR cycles for the same DNA template amounts.

taqman pcr assay conditions for ß-globin and c-erbb-2 genes
Using the fluorogenic probes for ß-globin and c-erbB-2 and the experimental conditions previously defined for the ß-actin gene, we obtained a linear relationship between the DNA concentration and the fluorescent signal ({Delta}RQ) for ß-globin and c-erbB-2 genes in 2.5–20 ng of DNA target. Even if the linear response for the two genes showed different slope coefficients (mainly attributable to differences in efficiencies of fluorescence-generating reactions), the c-erbB-2/ß-globin fluorescence ratio was constant in this range of target concentrations of normal DNAs (Fig. 4 ).



View larger version (20K):
[in this window]
[in a new window]
  		Figure 4. Linear relationships between amounts of DNA target and fluorescence output, expressed as {Delta}RQ, for ß-globin and c-erbB-2 genes.

A 30-cycle amplification was performed on 2.5, 5, 10, and 20 ng of a normal DNA. The dotted line indicates the stability of c-erbB-2/ß-globin ratio at various DNA doses.

measuring c-erbb-2 amplification in breast tumor specimens
To determine the amplification extent of c-erbB-2 oncogene in breast tumor specimens, we tested 2.5 ng each of ß-globin and c-erbB-2 in different tubes but in the same run. Four replicates of human placental DNA were also assayed for both ß-globin and c-erbB-2 genes. For each unknown sample, we determined the {Delta}RQ values for both genes as well as the c-erbB-2/ß-globin ratio. Finally, we divided this ratio by the mean c-erbB-2/ß-globin fluorescence ratio obtained in placental DNA samples. When c-erbB-2 amplification values were >10, the DNA sample being measured was diluted before the TaqMan PCR assay.

assay performance
To test the precision of TaqMan-based PCR assay, we measured in the same assay the {Delta}RQ values for c-erbB-2 in 10 replicates of normal DNA, starting with two different template amounts (2.5 and 8 ng). The intraassay CV of the {Delta}RQ values for c-erbB-2 in each template was 3.3% and 4.7%, respectively. The interassay precision in 6 different runs was 15.4% and 17.3%, respectively.

We tested the capacity of the TaqMan system to discriminate between different-fold c-erbB-2 amplifications by measuring the amplification in reconstituted samples obtained by serial dilutions in placental DNA of a DNA extracted from the SKBR.3 cell line carrying an 11-fold c-erbB-2 amplification (24). The results are reported in Fig. 5 .



View larger version (21K):
[in this window]
[in a new window]
  		Figure 5. Accuracy of the homogeneous PCR assay based on the TaqMan system.

Decreasing amounts of DNA extracted from SKBR.3 cell line (T), containing ~11- fold-amplified c-erbB-2 gene, were mixed with increasing amounts of placental DNA (N) in the following proportions of T:N: A, 4:0; B, 3:1; C, 2:2; D, 1:3; E, 0:4. Reconstituted samples were designed to have a constant amount of DNA in each sample (2.5 ng) but different numbers of c-erbB-2 gene copies (from 11 to 1). We tested these reconstituted samples and plotted the expected values for c-erbB-2 amplification against the values derived from the reconstitution experiment. Linear regression analysis of the results is shown.

To verify the correspondence with classical hybridization techniques, we quantified c-erbB-2 amplification by the proposed method in four breast cancer cell lines (SKBR-3, ZR75.1, T47D, and MDA.MB.231) in which the oncogene amplification had been previously measured with Southern transfer analysis (Table 2 ).


View this table:
[in this window]
[in a new window]
  		Table 2. c-erbB-2 amplification in human breast cancer cell lines as obtained with Southern transfer analysis and the TaqMan assay.

Finally, we determined the amplification of c-erbB-2 in DNA extracted from 25 formalin-fixed, paraffin-embedded breast tumor samples in which the oncogene amplification had already been documented by a competitive PCR assay. The results obtained with the two PCR-based techniques showed a good correlation: r = 0.94, P <0.001 (Fig. 6 ).



View larger version (26K):
[in this window]
[in a new window]
  		Figure 6. Linear relation of c-erbB-2 amplification in 25 breast cancers as measured by the TaqMan procedure and by the competitive PCR.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In breast cancer, several different oncogenes (c-erbB-2, c-myc, and int-2) have been reported as potentially amplified (25). The prognostic value of these molecular lesions has been widely examined. In particular, in most reports, the amplification of c-erbB-2 has been considered prognostically unfavorable, being related to reduced survival and shorter relapse-free intervals in breast and ovarian carcinoma (15)(16)(26)(27)(28)—although conflicting results have been reported (18)(29)(30)(31). These differences can be explained by the high variability of semiquantitative assay procedures used in such studies, based mainly on Southern transfer or dot–blot analysis. New techniques, performed with standardized and well-controlled protocols, may represent an improved approach for understanding the clinical relevance of this and other quantitative molecular lesions in human cancers.

Accurate quantitative procedures for measuring nucleic acids, combined with the high sensitivity of PCR, are new and suitable tools for addressing specific requirements of diagnostic investigations (as in determinations of oncogene amplification) and are now becoming available for use in the clinical laboratory (32). However, up-to-date, independent, quantitative PCR methods usually require cumbersome postamplification procedures that can be additional sources of inaccuracy and contamination.

The novel procedure reported here allows accurate determination of PCR-amplified targets by a homogeneous technique. The specific signal derived by the use of a fluorogenic probe accumulates during PCR cycling and can easily be measured by end-point evaluation of specific fluorescence. The only difference between this system and other routine PCR procedures is the addition of a single reagent to the reaction mixture: a fluorogenic probe designed to anneal to the target sequence. In this sense, this system provides simultaneously (a) the desired information for quantitative evaluation and (b) qualitative recognition of the amplified target. The presence of a specific internal probe, carrying the signal-generating system, guarantees the specificity of the PCR product to be measured. Further, the intensity of the fluorescence signal allows the detection of PCR products in samples originally containing low quantities of DNA templates, even after a limited number of PCR cycles.

Oncogene amplification is a common quantitative alteration in tumoral DNA; therefore, we chose the increase of c-erbB-2 oncogene copy number as a suitable model for developing a quantitative TaqMan PCR assay. We first assessed the PCR conditions that would yield a linear response between DNA target doses and fluorescence signal from amplified products—the preliminary requirement for assessing a quantitative assay in the exponential phase of PCR. Using ß-actin, ß-globin, and c-erbB-2 fluorescent probes, we determined the range of both the number of PCR cycles and the amounts of DNA that should be used for maintaining the linear relationship between amplified target and PCR-generated product. These preliminary data were then applied to the development of the assay for c-erbB-2 amplification. Results for DNA extracted from paraffin blocks of 25 breast primary tumors, which provided only limited quantities of variable degraded DNA unsuitable for Southern transfer analysis, were compared with those obtained with an already developed competitive PCR procedure. This latter method is known to provide highly accurate and sensitive results, but the procedure (competitor construction, assessment of competitive PCR conditions, and measurement of PCR products) is quite complex. However, the TaqMan PCR assay described here represents a promising approach to obtaining reasonable accuracy for a quantitative PCR assay. The TaqMan PCR assay gave results comparable with those of Southern transfer analysis in DNA extracted from four breast cancer cell lines.

We conclude that the TaqMan procedure is easily applicable to qualitative applications in its standard configuration but, when the PCR conditions are well standardized and the exponential phase is well controlled, can also be successfully extended to quantitative applications. Our results showed a good agreement between the TaqMan assay and competitive PCR for determining the extent of c-erbB-2 amplification. Further, evaluation of precision and accuracy seems to indicate reliability of the TaqMan procedure for this measurement, apparently opening further improvements for quantitative PCR methods. However, further methodological studies will be necessary to confirm that the TaqMan methodology can be considered an accurate and sensitive quantitative PCR methodology.


   Acknowledgments
 
We are grateful to Perkin-Elmer Italia for providing fluorogenic probes and instrumentation used in this study. This work was supported by a grant of the University of Florence (Italy).


   Footnotes
 
Clinical Biochemistry Unit, Department of Clinical Physiopathology, University of Florence, Viale Pieraccini 6, 50134, Florence, Italy.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Kemp DJ, Smith DB, Foots J, Samaras N, Peterson MG. Colorimetric detection of specific DNA segments amplified by polymerase chain reaction. Proc Natl Acad Sci U S A 1989;86:2423-2427. [Abstract/Free Full Text]
  2. Saiki RK, Walsh PS, Levenson CH, Erlich HA. Genetic analysis of amplified DNA with immobilized sequence-specific oligonucleotide probes. Proc Natl Acad Sci U S A 1989;86:6230-6234. [Abstract/Free Full Text]
  3. Lunderberg J, Wahlberg J, Uhlen M. Rapid colorimetric quantification of PCR-amplified DNA. BioTechniques 1991;10:68-75. [Web of Science][Medline] [Order article via Infotrieve]
  4. Musiani M, Zerbini M, Gibellini D, Gentiloni G, La Placa H, Ferri E, Girotti S. Chemiluminescent assay for the detection of viral and plasmid DNA using digoxigenin-labeled probes. Anal Biochem 1991;194:394-398. [Web of Science][Medline] [Order article via Infotrieve]
  5. Landgraf A, Reckmann B, Pingoud A. Quantitative analysis of polymerase chain reaction (PCR) products using primers labeled with biotin and a fluorescent dye. Anal Biochem 1991;193:231-235. [Web of Science][Medline] [Order article via Infotrieve]
  6. Hruza C, Dobianer K, Beck A, Czerwenka K, Hanak H, Klein M, et al. HER-2 and int-2 amplification estimated by quantitative PCR in paraffin embedded ovarian cancer tissue sample. Eur J Cancer 1993;29:1593-1597.
  7. Frye RA, Benz CC, Liu E. Detection of amplified oncogenes by differential polymerase chain reaction. Oncogene 1989;4:1153-1157. [Web of Science][Medline] [Order article via Infotrieve]
  8. Sestini R, Orlando C, Zentilin L, Gelmini S, Pinzani P, Bianchi S, et al. Measuring c-erbB-2 amplification in fresh and paraffin-embedded tumors by competitive polymerase chain reaction. Clin Chem 1994;40:630-636. [Abstract/Free Full Text]
  9. Sestini R, Orlando C, Zentilin L, Lami D, Gelmini S, Pinzani P, et al. Measurement of gene amplification for c-erbB-2, c-myc, epidermal growth factor receptor, int-2 and N-myc by quantitative PCR with a multiple competitor template. Clin Chem 1995;41:826-832. [Abstract/Free Full Text]
  10. Harlow SP, Stewart CC. Quantification of c-myc gene amplification by competitive PCR assay system. PCR Methods Appl 1983;3:163-168.
  11. Lawyer FC, Stoffel S, Saiki RK, Myambo KB, Drummond R, Gelfand DH. Isolation, characterization and expression in Escherichia coli of the DNA polymerase gene from the extreme thermophile Thermus aquaticus. J Biol Chem 1989;264:6427-6437. [Abstract/Free Full Text]
  12. Holland PM, Abramson RD, Watson R, Gelfand DH. Detection of specific polymerase chain reaction product by utilizing the 5' to 3' exonuclease activity of Thermus aquaticus. DNA polymerase. Proc Natl Acad Sci U S A 1991;88:7276-7280. [Abstract/Free Full Text]
  13. Lyamichev V, Brow MAD, Dahlberg JE. Structure-specific endonucleolytic cleavage of nucleic acids by eubacterial DNA polymerases. Science 1993;260:778-783. [Abstract/Free Full Text]
  14. Lee LG, Connel CR, Bloch W. Allelic discrimination by nick translation PCR with fluorogenic probes. Nucleic Acid Res 1993;21:3761-3766. [Abstract/Free Full Text]
  15. Slamon DJ, Clark GM, Wong SG, Levin WJ, Ulrich A. Human breast cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogene. Science 1987;253:177-182.
  16. Slamon DJ, Godolphin W, Jone LA, Holt JA, Wong SG, Keit DE, et al. Studies of the HER-2/neu proto-oncogene in human breast and ovarian cancer. Science 1989;244:702-712. [Abstract/Free Full Text]
  17. Paterson MC, Dietrich KD, Danyluk J, Paterson AHG, Less AW, Jamil N, et al. Correlation between c-erbB-2 amplification and risk of recurrent disease in node-negative breast cancer. Cancer Res 1991;51:556-567. [Abstract/Free Full Text]
  18. Clark GM, McGuire WL. Follow up study of HER-2/neu amplification in primary breast cancer. Cancer Res 1991;51:944-948. [Abstract/Free Full Text]
  19. Perkin-Elmer. TaqMan probe design, synthesis and purification. Foster City. CA: Applied Biosystems, 1995..
  20. Forster V Th. Zwischenmolekular energie-wanderung und fluoreszenz. Ann Phys (Leipzig) 1948;2:55–75..
  21. Livak K, Flood SJA, Marmaro J, Giusti W, Deetz K. Oligonucleotides with fluorescent dyes at opposite ends provide a quencher probe system useful for detecting PCR product and nucleic acid hybridization. PCR Methods Appl 1995;4:357-362. [Web of Science][Medline] [Order article via Infotrieve]
  22. Impraim CC, Saiki RK, Erlich HE, Teplitz RL. Analysis of DNA extracted from formalin-fixed, paraffin-embedded tissues by enzymatic amplification and hybridization with sequence-specific oligonucleotides. Biochem Biophys Res Commun 1987;142:710-716. [Web of Science][Medline] [Order article via Infotrieve]
  23. Sambrook J, Fritsch EF, Maniatis T. Molecular cloning 1989:9-16 Cold Spring Harbor Laboratory Press Cold Spring Harbor, NY. .
  24. Hynes NE, Geber HA, Saurer S, Groner B. Overexpression of the c-erbB-2 protein in human breast tumor cell lines. J Cell Biochem 1989;39:145-151.
  25. Wolman SR, Pauley RJ, Mohamed AN, Dawson PJ, Visscher DW, Sarkar FH. Genetic markers as prognostic indicators in breast cancer. Cancer 1992;70:1765-1774. [Web of Science][Medline] [Order article via Infotrieve]
  26. Borg A, Tandon AK, Siggurdsson H, Clark GM, Ferno M, Fuqua SAW, et al. HER-2/neu amplification predicts poor survival in node-positive breast cancer. Cancer Res 1990;50:4332-4337. [Abstract/Free Full Text]
  27. Lönn U, Lönn S, Nilsson B, Stenkvist B. Prognostic value of erbB-2 and myc amplification in breast cancer imprints. Cancer 1995;75:2681-2687. [Web of Science][Medline] [Order article via Infotrieve]
  28. Perren T. c-erbB-2 oncogene as a prognostic marker in breast cancer. Br J Cancer 1991;63:328-332. [Web of Science][Medline] [Order article via Infotrieve]
  29. Ali IU, Campbell G, Lidereau R, Callahan R. Lack of evidence for the prognostic significance of c-erbB-2 amplification in human breast carcinoma. Oncogene Res 1988;3:139-146. [Web of Science][Medline] [Order article via Infotrieve]
  30. Zhou DJ, Ahuja H, Cline MJ. Proto-oncogene abnormalities in human breast cancer: c-erbB-2 amplification does not correlate with recurrence of disease. Oncogene 1989;45:360-363.
  31. Heintz NH, Leslie KO, Rogers LA, Howard PL. Amplification of the c-erbB-2 oncogene and prognosis of breast adenocarcinoma. Arch Pathol Lab Med 1990;114:160-163. [Web of Science][Medline] [Order article via Infotrieve]
  32. Thibodeau SN. Recent applications of PCR in clinical laboratory medicine [Editorial]. Clin Chem 1994;40:681-682. [Free Full Text]
  33. Kraus MH, Popescu NC, Amsbaugh SC, King CR. Overexpression of the EGF receptor-related protoncogene c-erbB-2 in human mammary cell lines by different molecular mechanisms. EMBO J 1987;6:605-610. [Web of Science][Medline] [Order article via Infotrieve]



The following articles in journals at HighWire Press have cited this article:


Home page
 JCOHome page
T. Gotoh, H. Hosoi, T. Iehara, Y. Kuwahara, S. Osone, K. Tsuchiya, M. Ohira, A. Nakagawara, H. Kuroda, and T. Sugimoto
Prediction of MYCN Amplification in Neuroblastoma Using Serum DNA and Real-Time Quantitative Polymerase Chain Reaction
J. Clin. Oncol., August 1, 2005; 23(22): 5205 - 5210.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Chem.Home page
L. Soldati, D. Adamo, C. Bianchin, T. Arcidiacono, A. Terranegra, M. L. Bianchi, S. Mora, D. Cusi, and G. Vezzoli
Vitamin D Receptor mRNA Measured in Leukocytes with the TaqMan Fluorogenic Detection System: Effect of Calcitriol Administration
Clin. Chem., August 1, 2004; 50(8): 1315 - 1321.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
U. Woelfle, J. Cloos, G. Sauter, L. Riethdorf, F. Janicke, P. van Diest, R. Brakenhoff, and K. Pantel
Molecular Signature Associated with Bone Marrow Micrometastasis in Human Breast Cancer
Cancer Res., September 15, 2003; 63(18): 5679 - 5684.
[Abstract] [Full Text] [PDF]

Home page
 J. Clin. Microbiol.Home page
A. Garcia-Quintanilla, J. Gonzalez-Martin, G. Tudo, M. Espasa, and M. T. Jimenez de Anta
Simultaneous Identification of Mycobacterium Genus and Mycobacterium tuberculosis Complex in Clinical Samples by 5'-Exonuclease Fluorogenic PCR
J. Clin. Microbiol., December 1, 2002; 40(12): 4646 - 4651.
[Abstract] [Full Text] [PDF]

Home page
 Nucleic Acids ResHome page
I. M. Mackay, K. E. Arden, and A. Nitsche
Real-time PCR in virology
Nucleic Acids Res., March 15, 2002; 30(6): 1292 - 1305.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
F. C. Kischkel, D. A. Lawrence, A. Tinel, H. LeBlanc, A. Virmani, P. Schow, A. Gazdar, J. Blenis, D. Arnott, and A. Ashkenazi
Death Receptor Recruitment of Endogenous Caspase-10 and Apoptosis Initiation in the Absence of Caspase-8
J. Biol. Chem., November 30, 2001; 276(49): 46639 - 46646.
[Abstract] [Full Text] [PDF]

Home page
 Appl. Environ. Microbiol.Home page
S. Becker, P. Böger, R. Oehlmann, and A. Ernst
PCR Bias in Ecological Analysis: a Case Study for Quantitative Taq Nuclease Assays in Analyses of Microbial Communities
Appl. Envir. Microbiol., November 1, 2000; 66(11): 4945 - 4953.
[Abstract] [Full Text]

Home page
 BloodHome page
P. Willems, O. Verhagen, C. Segeren, P. Veenhuizen, J. Guikema, E. Wiemer, L. Groothuis, T. B.-d. Jong, H. Kok, A. Bloem, et al.
Consensus strategy to quantitate malignant cells in myeloma patients is validated in a multicenter study
Blood, July 1, 2000; 96(1): 63 - 70.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
D. C. Sgroi, S. Teng, G. Robinson, R. LeVangie, J. R. Hudson Jr., and A. G. Elkahloun
In Vivo Gene Expression Profile Analysis of Human Breast Cancer Progression
Cancer Res., November 1, 1999; 59(22): 5656 - 5661.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Chem.Home page
C. C. Raggi, M. L. Bagnoni, G. P. Tonini, M. Maggi, G. Vona, P. Pinzani, K. Mazzocco, B. De Bernardi, M. Pazzagli, and C. Orlando
Real-Time Quantitative PCR for the Measurement of MYCN Amplification in Human Neuroblastoma with the TaqMan Detection System
Clin. Chem., November 1, 1999; 45(11): 1918 - 1924.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Cancer Res.Home page
P.-W. Chiang, D. G. Beer, W.-L. Wei, M. B. Orringer, and D. M. Kurnit
Detection of erbB-2 Amplifications in Tumors and Sera from Esophageal Carcinoma Patients
Clin. Cancer Res., June 1, 1999; 5(6): 1381 - 1386.
[Abstract] [Full Text] [PDF]

Home page
 Appl. Environ. Microbiol.Home page
D.-M. Norton and C. A. Batt
Detection of Viable Listeria monocytogenes with a 5' Nuclease PCR Assay
Appl. Envir. Microbiol., May 1, 1999; 65(5): 2122 - 2127.
[Abstract] [Full Text]

Home page
 J. Histochem. Cytochem.Home page
G.J. Nuovo, R.J. Hohman, G.A. Nardone, and I.A. Nazarenko
In Situ Amplification Using Universal Energy Transfer-labeled Primers
J. Histochem. Cytochem., March 1, 1999; 47(3): 273 - 280.
[Abstract] [Full Text]

Home page
 BloodHome page
C. Gonzalez-Manchon, M. Fernandez-Pinel, E. G. Arias-Salgado, M. Ferrer, M.-V. Alvarez, S. Garcia-Munoz, M. S. Ayuso, and R. Parrilla
Molecular Genetic Analysis of a Compound Heterozygote for the Glycoprotein (GP) IIb Gene Associated With Glanzmann's Thrombasthenia: Disruption of the 674-687 Disulfide Bridge in GPIIb Prevents Surface Exposure of GPIIb-IIIa Complexes
Blood, February 1, 1999; 93(3): 866 - 875.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
M. Ferrer, J. Tao, G. Iruin, M. Sanchez-Ayuso, J. Gonzalez-Rodriguez, R. Parrilla, and C. Gonzalez-Manchon
Truncation of Glycoprotein (GP) IIIa (triangle  616-762) Prevents Complex Formation With GPIIb: Novel Mutation in Exon 11 of GPIIIa Associated With Thrombasthenia
Blood, December 15, 1998; 92(12): 4712 - 4720.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
S. Sundaresan, P. E. Roberts, K. L. King, M. X. Sliwkowski, and J. P. Mather
Biological Response to ErbB Ligands in Nontransformed Cell Lines Correlates with a Specific Pattern of Receptor Expression
Endocrinology, December 1, 1998; 139(12): 4756 - 4764.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Chem.Home page
T. Yajima, A. Yagihashi, H. Kameshima, D. Kobayashi, D. Furuya, K. Hirata, and N. Watanabe
Quantitative reverse transcription-PCR assay of the RNA component of human telomerase using the TaqMan fluorogenic detection system
Clin. Chem., December 1, 1998; 44(12): 2441 - 2445.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
Y. Hirayama, S. Sakamaki, T. Matsunaga, T. Kuga, H. Kuroda, T. Kusakabe, K. Sasaki, K. Fujikawa, J. Kato, K. Kogawa, et al.
Concentrations of Thrombopoietin in Bone Marrow in Normal Subjects and in Patients With Idiopathic Thrombocytopenic Purpura, Aplastic Anemia, and Essential Thrombocythemia Correlate With Its mRNA Expression of Bone Marrow Stromal Cells
Blood, July 1, 1998; 92(1): 46 - 52.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Chem.Home page
M. V. Bor, B. S. Sorensen, P. Rammer, and E. Nexo
Calibrated user-friendly reverse transcriptase-PCR assay: quantitation of epidermal growth factor receptor mRNA
Clin. Chem., June 1, 1998; 44(6): 1154 - 1160.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Chem.Home page
D. Whitcombe, J. Brownie, H. L. Gillard, D. McKechnie, J. Theaker, C. R. Newton, and S. Little
A homogeneous fluorescence assay for PCR amplicons: its application to real-time, single-tube genotyping
Clin. Chem., May 1, 1998; 44(5): 918 - 923.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (59)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Gelmini, S.
Right arrow Articles by Pazzagli, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Gelmini, S.
Right arrow Articles by Pazzagli, M.
Related Collections
Right arrow Molecular Diagnostics and Genetics

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS