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Abstract
Real-time reverse transcription followed by polymerase chain reaction (RT-PCR) is
the most suitable method for the detection and quantification of mRNA. It offers high
sensitivity, good reproducibility and a wide quantification range. Today, relative
expression is increasingly used, where the expression of a target gene is standardised
by a non-regulated reference gene. Several mathematical algorithms have been developed
to compute an expression ratio, based on real-time PCR efficiency and the crossing
point deviation of an unknown sample versus a control. But all published equations
and available models for the calculation of relative expression ratio allow only for
the determination of a single transcription difference between one control and one
sample. Therefore a new software tool was established, named REST (relative expression
software tool), which compares two groups, with up to 16 data points in a sample and
16 in a control group, for reference and up to four target genes. The mathematical
model used is based on the PCR efficiencies and the mean crossing point deviation
between the sample and control group. Subsequently, the expression ratio results of
the four investigated transcripts are tested for significance by a randomisation test.
Herein, development and application of REST is explained and the usefulness of relative
expression in real-time PCR using REST is discussed. The latest software version of
REST and examples for the correct use can be downloaded at http://www.wzw.tum.de/gene-quantification/.