[Use of AmpliSensor to quantitate gene expression in small amounts of samples: comparison with the quantitative RT-PCR method using CCD imaging system].
Quantitative PCR methods are potentially useful for determining levels of specific gene expression and gene dosage. Previously we developed a non-radioisotopic quantitative RT-PCR method by utilizing the PCR amplification kinetics and CCD image analyzer. Recently, based on the principle of fluorescence energy transfer, the AmpliSensor system that can quantitate the PCR products concurrent to amplification in a single reaction vessel has been described. Herein, we compared the results obtained by both methods. cDNAs were synthesized from 10 human endometrial biopsy specimens. Aliquots of cDNA were used for quantitation by gel/image analyzer or AmpliSensor assay system. For estimation of the initial amount of the template(I), regression equations of the form:y = I x Ex, where y is the amount of PCR products and x is the number of cycles, were fitted to the data in the linear portion of the semi-logarithmic graphs. The relative levels of beta-actin cDNA estimated by AmpliSensor assay system was in good agreement (r = 0.91) with those of the gel/image analyzer assay system, which is cumbersome, time-consuming and needs post-amplification procedures. The AmpliSensor assay is suitable for a quantitative PCR assay based on PCR kinetics, and is applicable for diagnostic testings.
Yokoi
H
H
Department of Clinical Molecular Biology, Faculty of Medicine, Kyoto University.
Nonoguchi
K
K
Kishishita
M
M
Iwai
M
M
Higashitsuji
H
H
Fujita
J
J
D000199:Actins / Q000235:genetics; D000818:Animals; D018076:DNA, Complementary / Q000032:analysis*; D015870:Gene Expression; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D012194:RNA-Directed DNA Polymerase
D000199:Actins; D018076:DNA, Complementary; D012194:RNA-Directed DNA Polymerase