Journal ArticleDOI
Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper--Excel-based tool using pair-wise correlations.
TLDR
The developed, and herein presented, software called BestKeeper determines the best suited standards, out of ten candidates, and combines them into an index, which can be compared with further target genes to decide, whether they are differentially expressed under an applied treatment.Abstract:
The stability of standard gene expression is an elementary prerequisite for internal standardisation of target gene expression data and many so called housekeeping genes with assumed stable expression can exhibit either upor down-regulation under some experimental conditions. The developed, and herein presented, software called BestKeeper determines the best suited standards, out of ten candidates, and combines them into an index. The index can be compared with further ten target genes to decide, whether they are differentially expressed under an applied treatment. All data processing is based on crossing points. The BestKeeper software tool was validated on four housekeeping genes and 10 members of the somatotropic axis differentially expressed in bovine corpora lutea total RNA. The BestKeeper application and necessary information about data processing and handling can be downloaded on http://www.wzw.tum.de/gene-quantification/bestkeeper.htmlread more
Citations
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Journal ArticleDOI
qBase relative quantification framework and software for management and automated analysis of real-time quantitative PCR data
TL;DR: Advanced and universally applicable models for relative quantification and inter-run calibration with proper error propagation along the entire calculation track are outlined in qBase, a free program for the management and automated analysis of qPCR data.
Journal ArticleDOI
Genome-Wide Identification and Testing of Superior Reference Genes for Transcript Normalization in Arabidopsis
TL;DR: Hundreds of Arabidopsis genes were found that outperform traditional reference genes in terms of expression stability throughout development and under a range of environmental conditions, and the developed PCR primers or hybridization probes for the novel reference genes will enable better normalization and quantification of transcript levels inArabidopsis in the future.
Journal ArticleDOI
Quantification of mRNA using real-time RT-PCR
TL;DR: A series of RT-qPCR protocols are described that illustrate the essential technical steps required to generate quantitative data that are reliable and reproducible in molecular medicine, biotechnology, microbiology and diagnostics.
Journal ArticleDOI
Real-time RT-PCR normalisation; strategies and considerations
TL;DR: This review discusses the relative merits of different normalisation strategies, suggests a method of validation that will enable the measurement of biologically meaningful results and discusses the popular practice of measuring an internal reference or housekeeping gene.
Journal ArticleDOI
Real-time PCR for mRNA quantitation.
Marisa L. Wong,Juan F. Medrano +1 more
TL;DR: This review discusses the key components of a real-time PCR experiment, including one-step or two-step PCR, absolute versus relative quantitation, mathematical models available for Relative quantitation and amplification efficiency calculations, types of normalization or data correction, and detection chemistries.
References
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TL;DR: This study enters into the particular topics of the relative quantification in real-time RT-PCR of a target gene transcript in comparison to a reference gene transcript and presents a new mathematical model that needs no calibration curve.
Journal ArticleDOI
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Jo Vandesompele,Katleen De Preter,Filip Pattyn,Bruce Poppe,Nadine Van Roy,Anne De Paepe,Franki Speleman +6 more
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Journal ArticleDOI
Relative expression software tool (REST©) for group-wise comparison and statistical analysis of relative expression results in real-time PCR
TL;DR: Development and application of REST is explained, the usefulness of relative expression in real-time PCR using REST is discussed and the mathematical model used is based on the PCR efficiencies and the mean crossing point deviation between the sample and control group.
Journal Article
A reagent for the single-step simultaneous isolation of RNA, DNA and proteins from cell and tissue samples.
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Journal ArticleDOI
Housekeeping genes as internal standards: use and limits.
Olivier Thellin,Willy Zorzi,Bernard Lakaye,B. de Borman,Bernard Coumans,Georges Hennen,Thierry Grisar,Ahmed Igout,Ernst Heinen +8 more
TL;DR: It is recalled that the commonly used internal standards can quantitatively vary in response to various factors and possible variations are illustrated using three experimental examples.