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|Author(s):||J P. Jakupciak; Nathan D. Gallant; A H. Smith; Matthew Becker; Alessandro Tona; Donald H. Atha;|
|Title:||Improved Methods and Standards for Telomerase Detection: Quantitative Histopathology Using Antibody Staining|
|Published:||October 01, 2009|
|Abstract:||The enzyme telomerase is expressed in 85% to 90% of all human cancers, but not in normal, non-stem cell somatic tissues at detectable levels. Clinical studies using indirect measurements, like telomerase activity assays, have demonstrated an association between telomerase activity and cells that are cancerous. Further, robust clinical assays for measuring telomerase activity in easily obtained body fluids would have great utility as non-invasive, cost-effective methods for the early detection of cancer. However, the use of telomerase as a biomarker has been hindered due to a lack of direct telomerase measurement methods and standards to normalize data collected across platforms. Standards and reference materials would significantly expedite the acceptance and translation of biomarkers, including telomerase, for expanded diagnostic use. To this end we are developing an in situ imaging method using a standard protein chip and antibody staining for the presence of telomerase. In the current study we have used 3-D confocal microscopy and fluorescently-labeled telomerase antibodies to quantify telomerase in cultured human tumor cells that exhibit different telomerase expression levels and human fibroblast cells as a control. These fluorescence measurements were compared to telomerase activity measured by RApidTRAP/PCR, hTERT mRNA RT-PCR, and flow cytometry. We have fabricated a multi-chamber chip that may have clinical applications as a platform for these immunohistochemistry measurements. The telomerase protein chip potentially serves as a reference to quantify telomerase concentration at a molecular level. This combination of measurements of telomerase by tissue staining, TRAP/PCR activity, mRNA expression, and flow cytometry will ensure a more complete quantification of telomerase levels in each of the respective tissues at various stages of cancer. We consider this to be the first report of a telomerase protein chip involved in biomarker quantification|
|Citation:||Biotechnic & Histochemistry|
|Pages:||pp. 195 - 206|
|Keywords:||biomarker quantitation,confocal microscopy,Immunohistochemistry Chip,standards,Telomerase activity|
|Research Areas:||Life Sciences Research|