John P. Jakupciak, Wendy Wang, Peter E

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Analytical Validation of Telomerase Activity for Cancer Early Detection John P. Jakupciak, Wendy Wang, Peter E. Barker, Sudhir Srivastava, Donald H. Atha The Journal of Molecular Diagnostics Volume 6, Issue 3, Pages 157-165 (August 2004) DOI: 10.1016/S1525-1578(10)60506-5 Copyright © 2004 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 1 Automated system for high-throughput analysis of telomerase activity. The system incorporates a MWG RoboSeq 4204 for automated sample handling of RNA/protein that has been prepared from cancer patients or normal controls. Telomerase activity is determined using a TRAP/PCR that has been modified for robotic handling of samples and reagents. TRAP/PCR products are then analyzed on the ABI 3100 multi-capillary electrophoresis instrument. Samples prepared on the RoboSeq 4204 are also used for rapid, real-time assay of HTERT mRNA analysis using the Luminex or the LightCycler. Part A reprinted from the Weill Medical College of Cornell University Education Center's online resource “PathNotes”, entitled “Neoplasia. Biological Characteristics of Benign and Malignant Neoplasms” with permission from Robert C. Mellors, M.D., Ph.D. The Journal of Molecular Diagnostics 2004 6, 157-165DOI: (10.1016/S1525-1578(10)60506-5) Copyright © 2004 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 2 Real-time amplification and probe-specific detection of hTERT RNA using the LightCycler. The y-axis represents the relative ratio of fluorescence between the reference channel and the sample channel. Curves labeled 1 to 11 represent dilutions of extracted total RNA from fresh cultured A549 cells. For clarity, sample triplicates are not shown. Curve 1, RNA detection from 7000 cells; curve 2, RNA detection from 3500 cells; curve 3, RNA detection from 1750 cells; curve 4, RNA detection from 875 cells, curve 5, detection from 219 cells; curve 6, detection from 55 cells; curve 7, detection from 175 cells, curve 8, detection from 110 cells; curve 9, detection from 70 cells; curve 10, detection from 9 cells; and curve 11, results of the no template control. Each 6.6 cycle numbers represents a 100-fold difference in concentration of PCR product. The Journal of Molecular Diagnostics 2004 6, 157-165DOI: (10.1016/S1525-1578(10)60506-5) Copyright © 2004 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 3 Real-time data from cancer cell lines. The y-axis represents the relative ratio of fluorescence between the reference channel and the sample channel. A and B: Typical results from different replicates (R1 and R2) and their crossover threshold (Ct) values. A: Quantification of mRNA from 7000 cell equivalents. B: Quantification of mRNA from 110 cell equivalents. A549 cell cultures were treated with the same isolation methods and analyzed for their abundance of hTERT mRNA. Ct values were compared for each experiment. As shown, precision in measurement is best at high cell equivalents/reaction (A). For practical purposes, cell concentrations between 110 and 9 cells were not significantly different. The Journal of Molecular Diagnostics 2004 6, 157-165DOI: (10.1016/S1525-1578(10)60506-5) Copyright © 2004 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 4 Capillary electrophoresis of TRAP/PCR products generated using the MWG robot. The TRAP/PCR assay was performed using fluorescent-labeled TS primer as described previously for the manual method and analyzed on the Applied Biosystems 310 CE. A: Electropherograms produced from separate TRAP/PCR reactions using increasing concentrations of telomerase (A549 cell extract). The extension products range from about 40 bp to about 200 bp with 6 bp repeats as previously described.17 In our capillary electrophoresis method we have used the fluorescently labeled TS primer. The traditional slab-gel method instead uses a non-covalent stain such as Sybr Green I. The non-covalent dye does not bind the primer-dimer with enough affinity to produce the high intensity primer-dimer bands/peaks which appear in the CE method using the covalent-labeled primer. We correct the total peak area of the TRAP ladder peaks by subtracting or not including the contribution of these primer-dimer peaks. B: TRAP/PCR CE analysis of SRM 1951A. Electropherograms from SRM 1951A are shown in comparison to the blank (no telomerase) and the positive control (A549 cells). SRM 1951A (undiluted) shows no telomerase activity as evidenced by the absence of extension products. The Journal of Molecular Diagnostics 2004 6, 157-165DOI: (10.1016/S1525-1578(10)60506-5) Copyright © 2004 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 5 Plot of the total extension product peak areas as a function of telomerase concentration. Areas were calculated from the electropherograms shown in Figure 4A (filled symbols). Data obtained from manual TRAP/PCR (open symbols) is shown for comparison. Error bars (± 1 SD) are based on repeatability measurements for each method. Analysis was performed using GenoTyper and Excel as previously described.17 The Journal of Molecular Diagnostics 2004 6, 157-165DOI: (10.1016/S1525-1578(10)60506-5) Copyright © 2004 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 6 Comparison of hTERT mRNA and telomerase activity. The relationship between the relative telomerase activity (bars) and the amount of cellular hTERT mRNA (filled symbols). The amount of cellular hTERT mRNA (copy number) was measured in the A549 cells in comparison to the telomerase activity (total peak area) obtained from automated TRAP/PCR measurements (Figure 5). The Journal of Molecular Diagnostics 2004 6, 157-165DOI: (10.1016/S1525-1578(10)60506-5) Copyright © 2004 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions