Quantitative real-time RT-PCR – a perspective

Abstract

The real-time reverse transcription polymerase chain reaction (RT-PCR) uses fluorescent reporter molecules to monitor the production of amplification products during each cycle of the PCR reaction. This combines the nucleic acid amplification and detection steps into one homogeneous assay and obviates the need for gel electrophoresis to detect amplification products. Use of appropriate chemistries and data analysis eliminates the need for Southern blotting or DNA sequencing for amplicon identification. Its simplicity, specificity and sensitivity, together with its potential for high throughput and the ongoing introduction of new chemistries, more reliable instrumentation and improved protocols, has made real-time RT-PCR the benchmark technology for the detection and/or comparison of RNA levels.

Detection of human mammaglobin mRNA in serial peripheral blood samples from patients with non-metastatic breast cancer is not predictive of disease recurrence

“…The detection of these cells may depend on the number of cancer cells present in the blood and also on the hMAM expression level by individual cells [42]. Recently, quantitative real-time fluorescence-based RT-PCR has become the ideal method for the detection of RNA [43]. Some studies have applied this methodology in the analysis of mammaglobin mRNA expression in peripheral blood samples of breast cancer patients and still reported low sensitivity [36,44].…”

Egr-1 binds the GnRH promoter to mediate the increase in gene expression by insulin

“…PCR conditions were optimized to generate > 95% PCR efficiency and only those reactions with between 95-105% efficiency were included in subsequent analyses. Relative differences in cDNA concentration between baseline and test conditions were then calculated using the comparative threshold cycle (Ct) method (Bustin et al, 2005). Briefly, for each sample, a ΔCT was calculated to normalize for the internal control using the equation: ΔCT= CT(gene)− CT(18S).…”

Selecting and validating reference genes for quantitative real-time PCR in Plutella xylostella (L.)

“…Gene expression analysis is increasingly important in exploring the potential functions of genes in various organisms. Quantitative real-time PCR (qRT-PCR) has rapidly become a robust method for gene expression studies due to its convenience and reproducibility (Bustin et al 2005). However, some factors, such as RNA stability, the variable efficiency of reverse transcription, and PCR efficiency, can affect its reliability.…”