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An exploratory investigation of various modes of action and potential adverse outcomes of fluoxetine in marine mussels

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      Analyzing real-time PCR data by the comparative CT method

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        Relative expression software tool (REST) for group-wise comparison and statistical analysis of relative expression results in real-time PCR.

        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/.
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          A new and rapid colorimetric determination of acetylcholinesterase activity

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            Author and article information

            Journal
            Aquatic Toxicology
            Aquatic Toxicology
            Elsevier BV
            0166445X
            June 2014
            June 2014
            : 151
            :
            : 14-26
            10.1016/j.aquatox.2013.11.016
            © 2014

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