Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 105, Issue 51, Pages 20173-20178Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.0806355106
Keywords
lab-on-a-chip; microfabrication; RNAi; stochastic gene expression
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Funding
- National Science Foundation
- National Institutes of Health Molecular Biophysics [T32GM08295]
- Physical Biosciences, Materials, and Chemical Sciences Divisions of the U.S. Department of Energy Divisions of the U.S. Department of Energy [DE-AC02-05CH11231]
- National Institutes of Health [HG003329]
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An integrated microdevice is developed for the analysis of gene expression in single cells. The system captures a single cell, transcribes and amplifies the mRNA, and quantitatively analyzes the products of interest. The key components of the microdevice include integrated nanoliter metering pumps, a 200-nL RT-PCR reactor with a single-cell capture pad, and an affinity capture matrix for the purification and concentration of products that is coupled to a microfabricated capillary electrophoresis separation channel for product analysis. Efficient microchip integration of these processes enables the sensitive and quantitative examination of gene expression variation at the single-cell level. This microdevice is used to measure siRNA knockdown of the GAPDH gene in individual Jurkat cells. Single-cell measurements suggests the presence of 2 distinct populations of cells with moderate (approximate to 50%) or complete (approximate to 0%) silencing. This stochastic variation in gene expression and silencing within single cells is masked by conventional bulk measurements.
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