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Specific Detection of Cytokeratin 20-Positive Cells in Blood of Colorectal and Breast Cancer Patients by a High Sensitivity Real-Time Reverse Transcriptase-Polymerase.

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Presentation on theme: "Specific Detection of Cytokeratin 20-Positive Cells in Blood of Colorectal and Breast Cancer Patients by a High Sensitivity Real-Time Reverse Transcriptase-Polymerase."— Presentation transcript:

1 Specific Detection of Cytokeratin 20-Positive Cells in Blood of Colorectal and Breast Cancer Patients by a High Sensitivity Real-Time Reverse Transcriptase-Polymerase Chain Reaction Method  Giuliana Giribaldi, Simone Procida, Daniela Ulliers, Franca Mannu, Roberta Volpatto, Giorgia Mandili, Laura Fanchini, Oscar Bertetto, Gianruggero Fronda, Luigi Simula, Elena Rimini, Giovanni Cherchi, Lisa Bonello, Milena Maria Maule, Francesco Turrini  The Journal of Molecular Diagnostics  Volume 8, Issue 1, Pages (February 2006) DOI: /jmoldx Copyright © 2006 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

2 Figure 1 CK20 amplification plots of serially diluted HT29 cells. Serially diluted HT29 cells (1, 104 cells; 2, 103 cells; 3, 102 cells; 4, 101 cells) amplified by S-PCR (A), 20N-PCR (B), and 35N-PCR (C). Numbers of cycles were plotted against fluorescence expressed as relative fluorescence units. The Journal of Molecular Diagnostics 2006 8, DOI: ( /jmoldx ) Copyright © 2006 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

3 Figure 2 Melting peak analysis of 20N-PCR products. The melting peaks result from plotting the negative first derivative of measured fluorescence emission (y axis) at a given temperature (x axis). Curves were obtained with pure HT29 cells (A and B); with CK20+ clinical samples (C); with intron-spanning primers E and G (A); and with primer E and exon-overlapping primer F (B and C). The Journal of Molecular Diagnostics 2006 8, DOI: ( /jmoldx ) Copyright © 2006 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

4 Figure 3 Nonspecific amplifications by conventional intron spanning primers. Amplification plots obtained with cDNA of lympho-monocytes from healthy donors (A); melting curves obtained from the same PCR reactions (B); gel electrophoresis analysis (2% agarose gel stained with ethidium bromide) of amplified material from lympho-monocytes from healthy donors (controls) and HT29 cells (C). The Journal of Molecular Diagnostics 2006 8, DOI: ( /jmoldx ) Copyright © 2006 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

5 Figure 4 Schematic positions of the intron-spanning primers E and G (A) and exon-overlapping primer F (B) used for N-PCR. The Journal of Molecular Diagnostics 2006 8, DOI: ( /jmoldx ) Copyright © 2006 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

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