REST 2005 is a new standalone software tool to estimate up and down-regulation for gene expression studies. The software addresses issues surrounding the measurement of uncertainty for expression ratios, by using randomisation and bootstrapping techniques. By increasing the number of iterations from 2,000 to 50,000 in this version hypothesis tests achieve a level of consistency on par withtraditional statistical tests. New confidence intervals for expression levels also allow scientists to measure not only the statistical significance of deviations, but also their likely magnitude, even in thepresence of outliers. Graphical output of the data via a whisker box-plots provide a visual representation of variation for each gene that highlights potential issues such as distribution skew.

=> download here:    SetupRest2005_1.9.12.zip

=>  download  =>  Manual  REST 2005 version 1.9.12

=>  Short description of REST-2005

Real-time PCR is being used increasingly as the method of choice for mRNA quantification, allowing rapid analysis of gene expression from low quantities of starting template. Despite a wide range of approaches, the same principles underlie all data analysis, with standard approaches broadly classiffed as either absolute or relative. In this study we use a variety of absolute and relative approaches of data analysis to investigate nocturnal c-fos expression in wild-type and retinally degenerate mice. In addition, we apply a simple algorithm to calculate the amplifcation effciency of every sample from its amplifcation profle. We confrm that nocturnal c-fos expression in the rodent eye originates from the photoreceptor layer, with around a 5-fold reduction in nocturnal c-fos expression in mice lacking rods and cones. Furthermore, we illustrate that differences in the results obtained from absolute and relative approaches are underpinned by differences in the calculated PCR effciency. By calculating the amplifcation effciency from the samples under analysis, comparable results may be obtained without the need for standard curves. We have automated this method to provide a means of streamlining the real-time PCR process, enabling analysis of experimental samples based upon their own reaction kinetics rather than those of artificial standards.

Download  DART  PCR version 1.0.xls   (Excel version)
Example_exp_data.xls   (Excel version)
Peirson DART version 1 (PDF)

DART-PCR provides a simple means of analysing real-time PCR data from raw flurescence data. This allows an automatic calculation of amplification kinetics, as well as performing the subsequent calculations for relative quantification and calculation of assay variability. Amplification efficiencies are also tested to dtect anomalus samples within groups (outlayers) and differences between experimatal groups (amplification equivalence).

---

Quantitative real-time PCR represents a highly sensitive and powerful technique for the quantitation of nucleic acids. It has a tremendous  potential for the high-throughput analysis of gene expression in research and routine diagnostics. However, the major hurdle is not the  practical performance of the experiments themselves but rather the efficient evaluation and the mathematical and statistical analysis of the  enormous amount of data gained by this technology, as these functions are not included in the software provided by the manufacturers of thedetection systems. In this work, we focus on the mathematical evaluation and analysis of the data generated by quantitative real-time PCR,  the calculation of the final results, the propagation of experimental variation of the measured values to the final results, and the statistical analysis. We developed a Microsoft® Excel®-based software application coded in Visual Basic for Applications, called Q-Gene, which  addresses these points. Q-Gene manages and expedites the planning, performance, and evaluation of quantitative real-time PCR experiments, as well as the mathematical and statistical analysis, storage, and graphical presentation of the data. The Q-Gene software application is a tool to cope with complex quantitative real-time PCR experiments at a high-throughput scale and considerably expedites and rationalizes the experimental setup, data analysis, and data management while ensuring highest reproducibility.

Quantitative real-time PCR represents a highly sensitive and powerful technique for the quantitation of nucleic acids. It has a tremendous potentialfor the high-throughput analysis of gene expression in research and routine diagnostics. However, the major hurdle is not the practical performance of the experiments themselves but rather the efficient evaluation and the mathematical and statistical analysis of the enormous amount of data gained by this technology, as these functions are not included in the software provided by themanufacturers of the detection systems. In this work, we focus on the mathematical evaluation and analysis of the data generated by quantitative real-time PCR, the calculation of the final results, the propagation of experimental variation of the measured values to the final results, and the statistical analysis. We developed a Microsoft Excel-based software application coded in Visual Basic for Applications, called Q-Gene, which addresses these points. Q-Gene manages and expedites the planning, performance, and evaluation and quantitative real-time PCR experiments, as well as the mathematical and statistical analysis, storage, and graphical presentation of the data. The Q-Gene software application is a tool to cope with complex quantitative real-time PCR experiments at a high-throughput scale and considerably expedites and rationalizes the experimental setup, data analysis, and data management while ensuring highest reproducibility.

Assumption-free analysis of quantitative real-time PCR data

Ramakers C, Ruijter JM, Deprez RH, Moorman AF. (2003)
Neurosci Lett  2003 Mar 13;339(1): 62-66

Department of Anatomy and Embryology K2-283, Experimental and Molecular
Cardiology Group, Academic Medical Centre, University of Amsterdam, Meibergdreef
15, 1105 AZ, Amsterdam, The Netherlands

Quantification of mRNAs using real-time polymerase chain reaction (PCR) by monitoring the product formation with the fluorescent dye SYBR Green I is being extensively used in neurosciences, developmental biology, and medical diagnostics. Most PCR data analysis procedures assume that the PCR efficiency for the amplicon of interest is constant or even, in the case of the comparative C(t) method, equal to 2. The latter method already leads to a 4-fold error when the PCR efficiencies vary over just a 0.04 range. PCR efficiencies of amplicons are usually calculated from standard curves based on either known RNA inputs or on dilution series of a reference cDNA sample. In this paper we show that the first approach can lead to PCR efficiencies that vary over a 0.2 range, whereas the second approach may be off by 0.26. Therefore, we propose linear regression on the Log(fluorescence) per cycle number data as an assumption-free method to calculate starting concentrations of mRNAs and PCR efficiencies for each sample.

LinRegPCR E-mail request link  =>  bioinfo@amc.uva.nl?subject=LinRegPCR

Download LinRegPCR help manual

---

Addressing fluorogenic real-time qPCR inhibition using the novel custom Excel file system 'FocusField2-6GallupqPCRSet-upTool-001' to attain consistently high fidelity qPCR reactions.

Jack M. Gallup and Mark R. Ackermann

Department of Veterinary Pathology, College of Veterinary Medicine, Iowa State University. Ames, Iowa 50011-1250. USA.

Biol. Proced. Online 2006;8:87-152.

The purpose of this manuscript is to discuss fluorogenic real-time quantitative polymerase chain reaction (qPCR) inhibition and to introduce/define a novel Microsoft Excel-based file system which provides a way to detect and avoid inhibition, and enables investigators to consistently design dynamically-sound, truly LOG-linear qPCR reactions very quickly. The qPCR problems this invention solves are universal to all qPCR reactions, and it performs all necessary qPCR set-up calculations in about 52 seconds (using a pentium 4 processor) for up to seven qPCR targets and seventy-two samples at a time – calculations that commonly take capable investigators days to finish. We have named this custom Excel-based file system "FocusField2- 6GallupqPCRSet-upTool-001" (FF2-6-001 qPCR set-up tool), and are in the process of transforming it into professional qPCR set-up software to be made available in 2007. The current prototype is already fully functional.

• Download  -  Gallup-excel file
• Download  -  Gallup_ppt_stratagene_seminar.zip
• Download  -  Gallup_entire_procedure.pdf
• Download  -  further talks by J.M. Gallup
  

SoFAR:  software for fully automatic evaluation of real-time PCR data.

Wilhelm J, Pingoud A, Hahn M.

Justus-Liebig-Universitat Giessen, Giessen, Germany.
Biotechniques. 2003 Feb;34(2):324-32

Wilhelm J, Pingoud A, Hahn M.
Anal Biochem. 2003 Jun 15;317(2):218-25.

Institut fur Biochemie, FB 08, Justus-Liebig-Universitat Giessen,
Heinrich-Buff-Ring 58, D-35392 Giessen, Germany.

Real-time quantitative polymerase chain reaction (PCR) with on-line fluorescence detection has become an important technique not only for determination of the absolute or relative copy number of nucleic acids but also for mutation detection, which is usually done by measuring melting curves. Optimum assay conditions have been established for a variety of targets and experimental setups, but only limited attention has been directed to data evaluation and validation of the results. In this work, algorithms for the processing of real-time PCR data are evaluated for several target sequences (p53, IGF-1, PAI-1, Factor VIIc) and compared to the results obtained by standard procedures. The algorithms are implemented in software called SoFAR, which allows fully automatic analysis of real-time PCR data obtained with a Roche LightCycler instrument. The software yields results with considerably increased precision and accuracy of quantifications. This is achieved mainly by the correction of phase of the signal curves. The melting curve data are corrected for signal changes not due to the melting process and are smoothed by fitting cubic splines. Therefore, sensitivity, resolution, and accuracy of melting curve analyses are improved.

---

http://www.metralabs.com/en/dindex.html

SoFAR^®is a software of biologists and medics for a quantitative analysis and interpretation of real time PCR measurements. Originally it was developed by Dr. Jochen Wilhelm for research on a precise quantifying of tumour suppressor genes and is now distributed and up-dated by MetraLabs^® GmbH exclusively. This software makes a fully automatic analysis and interpretation of measurement data possible, which are written down by LightCycler^® (Roche Diagnostics^®) or RapidCycler^® (Idaho Technology). To meet the highest demands of precision and safety in the analysis, robust algorithms were developed that guarantee reliable results even with suboptimal data. In combination with a thought through user friendly surface, real time PCR measureings are easy, fast and precise to analyse.

Complete analysis with just one mouse click

Simply open the file which is to analyse - no other steps are needed. Therefore one file is completely analysed with just one mouse click.

Accurate results

SoFAR controls automatically whether the criteria for a correct quantitative analysis are obliged. Included are automatic recognition and evaluation of the exponential phase of amplification curves as well as the calculated CT values. Curves which cannot be analysed correctly are marked.

Always best possible results

An efficient noise-filtering of the raw data of amplification and melting curves, makes more precise results possible. Independent signal changes from the amplification are automatically recognised and corrected. The automatic correction of temperature dependent quenches at melting curves also eliminates systematic errors and increases the sensitivity of a melting curve analysis.

Easy data export
 All results can be printed, saved or exported into other programmes as graphics or in tables. Extensive report functions make an exact documentation of all results easy. Diagrams which can be exported or copied in publishing quality can be changed and transformed in the layout from the user.