Quantitative analysis of polymerase chain reaction products using biotinylated dUTP incorporation.
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 beta-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-alpha, IL-1 beta, and purified porcine platelet-derived growth factor stimulations on ICAM-1 expression in smooth muscle cells.
Duplàa
C
C
Inserm Unité 8 de Cardiologie, Pessac, France.
Couffinhal
T
T
Labat
L
L
Moreau
C
C
Lamazière
J M
JM
Bonnet
J
J
D001483:Base Sequence; D001710:Biotin / Q000031:analogs & derivatives*; D015152:Blotting, Northern; D015815:Cell Adhesion Molecules / Q000235:genetics; D002478:Cells, Cultured; D004247:DNA / Q000235:genetics; D003856:Deoxyuracil Nucleotides; D005069:Evaluation Studies as Topic; D015870:Gene Expression; D006801:Humans; D018799:Intercellular Adhesion Molecule-1; D008969:Molecular Sequence Data; D009131:Muscle, Smooth, Vascular / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000706:statistics & numerical data; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D015815:Cell Adhesion Molecules; D003856:Deoxyuracil Nucleotides; D012333:RNA, Messenger; D018799:Intercellular Adhesion Molecule-1; D001710:Biotin; C075017:5-(N-(N-biotinyl-epsilon-aminocaproyl)-3-aminoallyl)-2'-deoxuridine 5'-triphosphate; D004247:DNA