Olivier Gruselle, Thierry Coche, Jamila Louahed

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Development of a Quantitative Real-Time RT-PCR Assay for the Detection of MAGE- A3–Positive Tumors Olivier Gruselle, Thierry Coche, Jamila Louahed The Journal of Molecular Diagnostics Volume 17, Issue 4, Pages 382-391 (July 2015) DOI: 10.1016/j.jmoldx.2015.03.008 Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 1 Alignment of melanoma antigen A (MAGE-A) gene family mRNA sequences for the design of PCR primers and dual hybridization probe for MAGE-A3. The dual hybridization probe is located in the middle of the amplicon. The oligonucleotide primers and dual hybridization probe were designed to hybridize in areas of greatest sequence divergence between MAGE-A3 and the other family members. The MAGE-A3 sequence is framed. Nucleotide differences compared with MAGE-A3 sequence are highlighted in gray. The Journal of Molecular Diagnostics 2015 17, 382-391DOI: (10.1016/j.jmoldx.2015.03.008) Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 2 Specificity of the melanoma antigen family A3 (MAGE-A3) RT-PCR primers within the MAGE-A family members in SYBR Green conditions without dual hybridization probe (A) or with quantitative real-time RT-PCR primers and dual hybridization probe (B). RT-PCR results are shown as the quantification cycle (Cq) values. All 10 MAGE-A family cDNAs publicly available at time of testing were measured in triplicate. Represented values are means ± SD. White bars indicate that the limit of the assay (40 Cq) was reached. Quantity of plasmids: a, 20 pg; b, 2 pg; c, 0.2 pg; d, 0.02 pg. The Journal of Molecular Diagnostics 2015 17, 382-391DOI: (10.1016/j.jmoldx.2015.03.008) Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 3 Linearity of the melanoma antigen family A3 (MAGE-A3) quantitative real-time RT-PCR assay. The linearity of the assay was assessed by using serial dilutions of RNA extracted from MZ2-MEL3.0 cells. Eight replicates of each dilution were tested (each represented by a circle), using the MAGE-A3 and β-actin primers and probes. RT-PCR results are expressed as quantification cycle (Cq) values. The Journal of Molecular Diagnostics 2015 17, 382-391DOI: (10.1016/j.jmoldx.2015.03.008) Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 4 Richard sigmoid curve showing the MZ2-MEL3.0 cell concentrations around the cutoff for which the result of the assay is uncertain. Fit, fitted curve; MAGE-A3, melanoma antigen family A3; Obs, observed values. The Journal of Molecular Diagnostics 2015 17, 382-391DOI: (10.1016/j.jmoldx.2015.03.008) Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 5 Tumor expression of MAGEA3 gene. The boxplots summarize the distribution of tumor expression values obtained by the quantitative real-time RT-PCR (RT-qPCR) assay related to the five different classes defined for the semiquantitative RT-PCR assay. Each box portion illustrates the first and the third quartiles per class, with the median value depicted as a vertical line through the box. Minimal and maximal values are represented by the left and right horizontal extension lines. In addition, for each class, the individual values are plotted. The vertical dotted line represents the cutoff of the RT-qPCR assay. The Journal of Molecular Diagnostics 2015 17, 382-391DOI: (10.1016/j.jmoldx.2015.03.008) Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions