Quantitative high-throughput measurement of gene expression with sub-zeptomole sensitivity by capillary electrophoresis
| Autor: | K. Leslie Powell, Howard D. Thames, Nikki Liburd, Lea Spyres, Stacey Arantes, Michael C. MacLeod, Ella Bedford, Earl F. Walborg, Mahmoud Rouabhia, C. Marcelo Aldaz, David L. Mitchell, Sally Gaddis |
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| Rok vydání: | 2005 |
| Předmět: |
Keratinocytes
Biophysics Gene Expression Bronchi Respiratory Mucosa Biology Sensitivity and Specificity Biochemistry Genome Cell Line Capillary electrophoresis Gene expression Humans RNA Messenger Mammary Glands Human Molecular Biology Gene Detection limit Messenger RNA Electrophoresis Capillary Epithelial Cells Cell Biology Fibroblasts Genes p53 Fluorescence Molecular biology Orders of magnitude (mass) Female |
| Zdroj: | Analytical Biochemistry. 345:284-295 |
| ISSN: | 0003-2697 |
| Popis: | Microarray technologies have provided the ability to monitor the expression of whole genomes rapidly. However, concerns persist with regard to quantitation and reproducibility, and the detection limits for individual genes in particular arrays are generally unknown. This article describes a semiautomated PCR-based technology, Q-RAGE, which rapidly provides measurements of mRNA abundance with extremely high sensitivity using fluorescent detection of specific products separated by capillary electrophoresis. A linear relationship between template concentration and fluorescent signal can be demonstrated down to template concentrations in the low aM region, corresponding to approximately 0.04 zmol (24 molecules) per reaction. The technique is shown to be quantitative over five orders of magnitude of template concentration, and average mRNA abundances of approximately 0.01 molecule per cell can be detected. A single predefined set of 320 primers provides 90–95% coverage of all eukaryotic genomes. Analysis of a set of 19 p53-regulated genes in untreated cultures of normal human epithelial cells, derived from three different tissues, revealed a 600-fold range of apparent constitutive expression levels. For most of the genes assayed, good correlations were observed among the expression levels in normal mammary, bronchial, and epidermal epithelial cells. |
| Databáze: | OpenAIRE |
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