Analytical Detection of Immunoglobulin Heavy Chain Gene Rearrangements in Gastric Lymphoid Infiltrates by Peak Area Analysis of the Melting Curve in the.

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Presentation transcript:

Analytical Detection of Immunoglobulin Heavy Chain Gene Rearrangements in Gastric Lymphoid Infiltrates by Peak Area Analysis of the Melting Curve in the LightCycler System Eduardo Retamales, Luis Rodriguez, Leda Guzman, Francisco Aguayo, Mariana Palma, Claudia Backhouse, Jorge Argandona, Erick Riquelme, Alejandro Corvalan The Journal of Molecular Diagnostics Volume 9, Issue 3, Pages 351-357 (July 2007) DOI: 10.2353/jmoldx.2007.050129 Copyright © 2007 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 1 Amount of monoclonal IgH gene rearrangement by melting curve and one-peak area analysis in the LightCycler System. A scale from 0.01 to 100 ng of DNA from the lymphoblastoid B-cell line LM was diluted into genomic DNA extracted from human tonsil and amplified by seminested protocol. The Journal of Molecular Diagnostics 2007 9, 351-357DOI: (10.2353/jmoldx.2007.050129) Copyright © 2007 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 2 One-peak area analysis of monoclonal IgH gene rearrangement after melting curve analysis in the LightCycler System. A: Example of gastric MALT lymphoma case (case 5 in Table 1). B: Example of chronic gastritis case (case 20 in Table 1). The peak areas command with one-peak area was chosen to calculate the area under the curve restricted to the largest peak present. x axis indicates temperature (°C); y axis indicates plotted of the negative first derivative of the fluorescence versus temperature (−dF/dT). Blue line is the melting curve. Red line is one-peak area analysis. The Journal of Molecular Diagnostics 2007 9, 351-357DOI: (10.2353/jmoldx.2007.050129) Copyright © 2007 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 3 Polyacrylamide gel electrophoresis analysis and ethidium bromide staining of monoclonal IgH gene rearrangement after seminested amplification in the LightCycler System. A: Examples of gastric MALT lymphoma case showing one band consistent with monoclonal IgH gene rearrangement (lanes 1 to 6). B: Examples of chronic gastritis cases showing a smear of multiple bands corresponding to polyclonal IgH gene rearrangement (lanes 1 to 6). MW, molecular weight marker (100 bp; Invitrogen). The Journal of Molecular Diagnostics 2007 9, 351-357DOI: (10.2353/jmoldx.2007.050129) Copyright © 2007 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 4 Detection of the minimum percentage of monoclonal IgH gene rearrangement by melting curve and one-peak area analysis. DNA from a gastric MALT lymphoma case (case 4) was diluted 50, 12.5, 6.25, 3.12, and 1.56% in a chronic gastritis case (case 14). The one-peak area analysis was 4.30 for undiluted, 4.64 for 50%, 4.25 for 25%, 4.64 for 12.5%, 3.98 for 6.25%, 4.58 for 3.21%, and 4.44 for 1.56% dilution of gastric MALT lymphoma in chronic gastritis case. The one-peak area analysis was 6.60 for the chronic gastritis case. The Journal of Molecular Diagnostics 2007 9, 351-357DOI: (10.2353/jmoldx.2007.050129) Copyright © 2007 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions