REST 2009

version 1  released  22. December 2009

REST 2009 Software is a standalone software tool, developed by M. Pfaffl (Technical University Munich) and QIAGEN, for analysis of gene expression data from quantitative real-time PCR experiments. Click the "Resources" tab to download REST 2009 Software free of charge.
Download newest version REST 2009  =>
Qiagen download page  via  Analysis Software

Download User Guide  =>  REST 2009 Software User Guide - English (PDF)

REST 2009 Software
For gene expression analysis using real-time PCR data from the Rotor-Gene Q and other cyclers
  • Estimates up and down regulation for gene expression studies
  • Analysis using randomization and bootstrapping techniques
  • Graphical data output via whisker-box plots
REST 2009 Software applies a mathematic model that takes into account the different PCR efficiencies of the gene of interest and reference genes. Compared to using a single reference gene, using multiple reference genes for normalization can improve the reliability of results.

Traditional relative quantitation enables estimation of gene expression. However, this method does not provide statistical information that is suitable for comparing expression in groups of treated and untreated samples in a robust manner. The integrated randomization and bootstrapping methods used in REST 2009 Software test the statistical significance of calculated expression ratios and can be used even when the data includes outliers. REST 2009 Software provides additional features for convenient and robust data analysis (see table "Convenient and robust data analysis").

Convenient and robust data analysis

Feature Description
REST RG mode An optional input method allows users to copy and paste results from a Rotor-Gene Q comparative quantitation analysis rather than importing standard curve and CT results.
Whisker-box plots export Expression variation for each gene is visualized in a whisker-box plot to highlight potential issues, such as a distribution skew. Whisker-box plots are exported by right-clicking the graph.
Improved randomization Randomization algorithms have been improved for better confidence intervals and more accurate p values.
Handling of standard curve variation Improvements have been made to the calculation of confidence intervals and p values. Efficiency is determined using the best fit for the standard curve and is used in the randomization process.

Applications REST 2009 Software is suitable for gene expression analysis using real-time PCR data from the Rotor-Gene Q and other cyclers.

Cited references

  1. Pfaffl, M.W. (2001) A new mathematical model for relative quantification in real-time RT-PCR. NAR 29, e45
  2. Pfaffl, M.W., Horgan, G.W., Dempfle, L. (2002) Relative expression software tool (REST) for group-wise comparison and statistical analysis of relative expression results in real-time PCR. NAR 30, e36
  3. Vandesompele J. et. al. (2002) Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol. 3, research0034.1
  4. Relative Expression Software Tool
  5. Randomization Tests
  6. Introduction to Randomization Tests
  7. Resampling Statistics: Randomization and the Bootstrap
  8. Quartiles and Box and Whisker Plots
REST 2009 Software is intended for molecular biology applications. This software is neither intended for the diagnosis, prevention, or treatment of a disease, nor has it been validated for such use either alone or in combination with other products. Therefore, the performance characteristics of the product for clinical use (i.e., diagnostic, prognostic, therapeutic, or blood banking) are unknown.

Alernative download link

REST 2009 for 64bit Windows

REST 2009 is a popular bioinformatics tool to analyze relevant gene expression changes from crossing points (CP) of amplification curves gathered by a Real-Time PCR (qPCR) experiment. REST was developed by Michael W. Pfaffl (Technical University Munich) and QIAGEN.

However, I found REST 2009 (v2.0.13) to cease working when moving from a 32bit Windows operating system (Windows XP) to a 64bit Windows operating system (Windows 7). The accepted solution until now was, to install a VirtualPC software including a Windows XP image optimized to run under Windows 7 (dubbed Windows XP Mode). Resourcewise it was a disaster, not to mention the hassle of backing up an entire second system. Also, the need for additional security software in your virtual image made the system even slower, remember Microsoft ceased to provide security updates for Windows XP from the 8th of April 2014. Occasionally your would need need to install other 3rd party software to complete your analyses, in my case, I needed to install a PDF printer to be able to save my REST 2009 reports in a standardazied way. On top of all that, you always need to cope with net drive assignments, virtual net drives that will provide access to the underlying OS from inside your virtual XP and that behave a lot different in XP than Windows 7, as well as some restrictions regarding the virtual desktop or virtual windows the VirtualPC software provides. For example, you won't be able to move the emulated REST 2009 windows to a second monitor.

After all this complaining about how bad this free functionality in Windows 7 is, I want to present my workaround that has since then worked very well for me.

Downloads needed

Patching REST 2009

The problem with REST 2009, which was most probably written in .NET is, that the author missed to flag the code as 32bit code. What happens, when executing undefined code under a 64bit system is, that the code is executed as 64bit and thus fails. Under a 32bit system, the 32bit code works fine.

Using CorFlags.exe, it is possible to subsequently edit those header flags, making a 64bit Windows able to correctly identify and interpret the code.

After the above mentioned software components are installed, CorFlags.exe should reside in 
%PROGRAMFILES%\Microsoft SDKs\Windows\v6.1\bin 

whereas Rest.exe is located at 

Using an elevated command prompt (hit the WINDOWS key, search for cmd.exe, right-click cmd.exe and select "Run as administrator..."), simply enter the following command (or paste the code using the right mouse button): 

cmd.exe /c ""%PROGRAMFILES%\Microsoft SDKs\Windows\v6.1\bin\CorFlags.exe" "%PROGRAMFILES(X86)%\REST 2009\Rest.exe" "/32bit+"" & pause 

If there is no error message appearing in your command prompt, everything worked as expected and you might try running the Rest.exe directly under your 64bit Windows installation.

This instruction is copyright by Christian Wolf (2015) and may not be reproduced without written permission, instead you may freely link to this page to help others coping with the same problem. Last updated: 27.05.2015