title: Electrochemical detection of gene expression in tumor samples: overexpression of Rak nuclear tyrosine kinase.
abstract: Absolute quantification of Rak nuclear tyrosine kinase mRNA in breast tissue samples was determined by competitive RT-PCR. The total RNA from the same samples was also chemically amplified through conventional RT-PCR, and the relative amounts of these amplified RT-PCR products were determined by adsorption onto an indium tin oxide (ITO) electrode followed by electrochemical detection. The electrochemical detection was performed using the inorganic metal complex Ru(bpy)(3)(2+) (bpy = 2,2' bipyridine) to catalyze the oxidation of the guanine residues of the immobilized RT-PCR products. Using the competitive RT-PCR values as standards, it was found that an optimized conventional RT-PCR coupled with electrochemical detection provides a simple method for measuring relative gene expression among a series of mRNA samples from breast tumors. The use of electrochemical detection potentially eliminates the need for gel electrophoresis and fluorescent or radioactive labels in detecting the target genes.
authors.affiliation: Department of Chemistry and Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-3290, USA.
authors.forename: Paul M
authors.initials: PM
authors.lastname: Armistead
authors.forename: H Holden
authors.initials: HH
authors.lastname: Thorp
mesh_terms: D001483:Base Sequence; D001943:Breast Neoplasms / Q000201:enzymology / Q000235:genetics*; D004563:Electrochemistry / Q000379:methods*; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D009363:Neoplasm Proteins; D011505:Protein-Tyrosine Kinases / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012334:RNA, Neoplasm / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D009363:Neoplasm Proteins; D012333:RNA, Messenger; D012334:RNA, Neoplasm; D011505:Protein-Tyrosine Kinases; C092830:FRK protein, human