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Quantitative Analysis of Polymerase Chain Reaction Products Using Biotinylated dUTP Incorporation

Authors
Journal
Analytical Biochemistry
0003-2697
Publisher
Elsevier
Publication Date
Volume
212
Issue
1
Identifiers
DOI: 10.1006/abio.1993.1316

Abstract

Abstract A method for relative quantitation of specific mRNA species by polymerase chain reaction (PCR) has been developed by using the incorporation of biotinylated dUTP. Transferred biotinylated PCR products gave a sensitive colorimetric signal which could be quantitated by video analysis. In the exponential phase of amplification, the linearity and reproducibility of reverse transcription and PCR demonstrated the same efficiency of cDNA synthesis and PCR for the two target genes, ICAM-1 and β-actin, and allowed the normalization of ICAM-1 expression. These results suggested that in the exponential phase of amplification a relative quantitation of mRNA could be determined. We used this approach to analyze the different effects of TNF-α, IL-1β, and purified porcine platelet-derived growth factor stimulations on ICAM-1 expression in smooth muscle cells.

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