Quantitative Analysis of Polymerase Chain Reaction Products Using Biotinylated dUTP Incorporation

• Published in: Analytical biochemistry Vol. 212; no. 1; pp. 229 - 236
• Main Authors: Duplaa, C., Couffinhal, T., Labat, L., Moreau, C., Lamaziere, J.M.D., Bonnet, J.
• Format: Journal Article
• Language: English
• Published: San Diego, CA Elsevier Inc 01.07.1993; Elsevier
• Subjects: Base Sequence; Biological and medical sciences; Biotin - analogs & derivatives; Blotting, Northern; Cell Adhesion Molecules - genetics; Cells, Cultured; Deoxyuracil Nucleotides; Diverse techniques; DNA - genetics; Evaluation Studies as Topic; Fundamental and applied biological sciences. Psychology; Gene Expression; Humans; Intercellular Adhesion Molecule-1; Molecular and cellular biology; Molecular Sequence Data; Muscle, Smooth, Vascular - metabolism; Polymerase Chain Reaction - methods; Polymerase Chain Reaction - statistics & numerical data; Reproducibility of Results; RNA, Messenger - analysis; RNA, Messenger - genetics; Sensitivity and Specificity; Human; Cell culture; Induction; Reaction product; Cytokine; Smooth muscle; Colorimetry; Chemical labelling; Gene expression; Biotine; Uracil nucleotide; Polymerase chain reaction; Messenger RNA; Complementary DNA; Gene; Investigation method; Quantitative analysis
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1006/abio.1993.1316

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.