Quantitative high-throughput measurement of gene expression with sub-zeptomole sensitivity by capillary electrophoresis.
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.
Spyres
Lea
L
Department of Carcinogenesis, University of Texas M. D. Anderson Cancer Center, Smithville, TX 78957, USA.
Gaddis
Sally
S
Bedford
Ella
E
Arantes
Stacey
S
Liburd
Nikki
N
Powell
K Leslie
KL
Thames
Howard
H
Mitchell
David
D
Walborg
Earl
E
Rouabhia
Mahmoud
M
Aldaz
C Marcelo
CM
MacLeod
Michael C
MC
D001980:Bronchi / Q000166:cytology; D002460:Cell Line; D019075:Electrophoresis, Capillary; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism; D005260:Female; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D015870:Gene Expression; D016158:Genes, p53; D006801:Humans; D015603:Keratinocytes / Q000166:cytology / Q000378:metabolism; D042361:Mammary Glands, Human / Q000166:cytology / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D020545:Respiratory Mucosa / Q000166:cytology / Q000378:metabolism; D012680:Sensitivity and Specificity
D012333:RNA, Messenger