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(American Journal of Pathology. 2004;164:795-806.)
© 2004 American Society for Investigative Pathology

Technical Advance

Single-Channel Quantitative Multiplex Reverse Transcriptase-Polymerase Chain Reaction for Large Numbers of Gene Products Differentiates Nondemented from Neuropathological Alzheimer’s Disease

Stavros Therianos, Min Zhu, Eunice Pyun and Paul D. Coleman

From the Center for Aging and Developmental Biology, University of Rochester Medical Center, Rochester, New York

Effective approaches using array technologies are critical to understand the molecular bases of human diseases. The results obtained using such procedures require analysis and validation procedures that are still under development. In the context of Alzheimer’s disease, in which the identification of molecular mechanisms of underlying pathologies is vital, we describe a robust assay that is the first real-time reverse transcriptase-polymerase chain reaction-based high-throughput approach that can simultaneously quantitate the expression of a large number of genes at the copy number level from a minute amount of starting material. Using this approach within the human brain, we were able to quantitate as many as 19 genes at a time with only one type of fluorescent probe. The number of genes included can be considerably increased. Examples of consistent changes in Alzheimer’s disease within these 19 candidate genes included reductions in targets related to the dendritic and synaptic apparatus. These changes were specific to Alzheimer’s disease when compared with Parkinson’s disease cases. We also present comparison data with microarray analysis from the same brain region and the same patients. The high sensitivity and reproducibility of this technology coupled with appropriate multivariate analysis is proposed here to form a biotechnology platform that can be widely used for diagnostic purposes as well as basic research.






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Copyright © 2004 by the American Society for Investigative Pathology.