Real time quantitative PCR.

Abstract

We have developed a novel "real time" quantitative PCR method. The method measures PCR product accumulation through a dual-labeled fluorogenic probe {i.e., TaqMan Probe}. This method provides very accurate and reproducible quantitation of gene copies. Unlike other quantitative PCR methods, real-time PCR does not require post-PCR sample handling, preventing potential PCR product carry-over contamination and resulting in much faster and higher throughput assays. The real-time PCR method has a very large dynamic range of starting target molecule determination (at least five orders of magnitude}. Real-time quantitative PCR is extremely accurate and less labor-intensive than current quantitative PCR methods.

Proteasome inhibitor induced gene expression profiles reveal overexpression of transcriptional regulators ATF3, GADD153 and MAD1

“…In addition, the regulation of poorly expressed genes might be overestimated on the cDNA microarray chips. Development of the quantitative real-time RT ± PCR method for the determination of relative changes in gene expression o ers the added advantage of expanding this approach beyond simple pair-wise comparisons (Heid et al, 1996). The application of cDNA array hybridization as a high-throughput screening method for di erential gene expression has been applied to develop a more comprehensive picture of pathways associated with genotoxic stress (Amundson et al, 1999) and to characterize transformation related genes in oral cancer cells (Chang et al, 1998) and in ®broblasts (Tchernitsa et al, 1999).…”

HIV-1-derived lentiviral vectors and fetal route of administration on transgene biodistribution and expression in rhesus monkeys

“…Realtime PCR assays were performed on the ABI PRISM 7700 s Sequence Detection System as reported previously. 43,44 To determine the percentage of transduced cells in tissues, the calculated number of EGFP copies was normalized to e-globin as an internal control in order to reduce the variability introduced by processing and PCR. The low level of sensitivity of the assay is five copies of the EGFP transgene.…”

Loss of Membranous Expression of β-Catenin Is Associated with Tumor Progression in Cutaneous Melanoma and Rarely Caused by Exon 3 Mutations

“…The parameter Ct (threshold cycle) is defined as the fractional cycle number at which the fluorescence generated by cleavage of the probe passes a fixed threshold above baseline (18). The ␤-catenin target gene copy number in unknown samples is quantified by measuring Ct and by using a standard curve to determine the starting copy number.…”