TY - JOUR
T1 - Quantitative high-throughput measurement of gene expression with sub-zeptomole sensitivity by capillary electrophoresis
AU - Spyres, Lea
AU - Gaddis, Sally
AU - Bedford, Ella
AU - Arantes, Stacey
AU - Liburd, Nikki
AU - Powell, K. Leslie
AU - Thames, Howard
AU - Mitchell, David
AU - Walborg, Earl
AU - Rouabhia, Mahmoud
AU - Aldaz, C. Marcelo
AU - MacLeod, Michael C.
N1 - Funding Information:
This work was supported by grants CA84978 and CA09480 from the National Cancer Institute; grants ES11047, ES07784, and ES11235 from the National Institute of Environmental Health Sciences; and the University of Texas M. D. Anderson Cancer Center Tobacco Program. We thank Capital Genomix for sharing information related to the PureBind bead technique from its GeneSystem320 protocol. We appreciate the expert technical assistance of Rebecca Deen and Joi Holcomb in manuscript preparation.
PY - 2005/10/15
Y1 - 2005/10/15
N2 - 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.
AB - 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.
KW - Capillary electrophoresis
KW - Gene expression analysis
KW - PCR
KW - p53-Regulated gene
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U2 - 10.1016/j.ab.2005.07.022
DO - 10.1016/j.ab.2005.07.022
M3 - Article
C2 - 16125665
AN - SCOPUS:25444469673
SN - 0003-2697
VL - 345
SP - 284
EP - 295
JO - Analytical Biochemistry
JF - Analytical Biochemistry
IS - 2
ER -