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

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/.

Incremental expression of Tlr4 correlates with mouse resistance to Salmonella infection and fine regulation of relevant immune genes

“…44 The software REST 384 (http://www. gene-quantification.info/) 45 was used to analyze the relative expression of our measured transcripts using an efficiency corrected ratio. Error estimations of the calculated ratios were obtained using a Taylor's series as implemented in REST.…”

The knockdown of maternal glucocorticoid receptor mRNA alters embryo development in zebrafish

“…All analyses were performed in triplicate. The Relative Expression Software Tool 2009 (Pfaffl et al, 2002) (REST 2009) was used to estimate relative fold changes in the genes of interest, using a ratio of the Ct values and the PCR amplification efficiencies of the genes of interest and the housekeeping gene. REST 2009 uses randomization and bootstrapping methods to test the statistical significance of the gene expression ratios and calculate 95% confidence intervals for relative fold changes (Pfaffl, 2009).…”

Custom Design of a GeXP Multiplexed Assay Used to Assess Expression Profiles of Inflammatory Gene Targets in Normal Colon, Polyp, and Tumor Tissue

“…22,25,26,33 Thus, it is not unexpected that the three technology platforms yield slight variations in significantly altered genes.…”