Electrochemical detection of gene expression in tumor samples: overexpression of Rak nuclear tyrosine kinase.
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.
Armistead
Paul M
PM
Department of Chemistry and Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-3290, USA.
Thorp
H Holden
HH
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
D009363:Neoplasm Proteins; D012333:RNA, Messenger; D012334:RNA, Neoplasm; D011505:Protein-Tyrosine Kinases; C092830:FRK protein, human