© Dr. Michael W. Pfaffl email@example.com
and summarize all technical aspects involved in quantitative
gene expression analysis using real-time PCR & RT-PCR.
( specificity, sensitivity, reproducibility, intra- & inter-assay variations,
kinetic PCR efficiency calculation, optimization strategy, etc.)
The GENE QUANTIFICATION web page illustrates the usefulness of a reliable quantification strategy, and the difference between absolute vs. relative quantification assays in kinetic PCR & kinetic RT-PCR.
RT-PCR is the technique of choice for analyzing
mRNA in extremely low abundance. Real-time RT-PCR using SYBR Green I detection
combines the ease and necessary exactness to be able to produce reliable
as well as rapid results.
The reverse transcription - polymerase chain reaction (RT-PCR) is the most sensitive method for the detection of low abundance mRNA, often obtained from limited tissue samples. However, real-time RT-PCR is a complex technique, there are substantial problems associated with its true sensitivity, reproducibility and specificity and, as a quantitative method, it suffers from the problems inherent in RT and PCR. The recent introduction of fluorescence based kinetic (real-time) RT-PCR procedures significantly simplifies the process of producing reproducible quantification of mRNAs and promises to overcome these limitations. Nevertheless, their successful application depends on a clear understanding of the practical problems, and careful experimental design, application and validation remain essential for accurate quantitative measurements of the transcriptom.
optimized for Netscape
Navigator v4.7 and for Internet Explorer v5.0 (and higher)