Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper – Excel-based tool using pair-wise correlations

@article{Pfaffl2004DeterminationOS,
  title={Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper – Excel-based tool using pair-wise correlations},
  author={Michael W. Pfaffl and Ale{\vs} Tichop{\'a}d and Christian Prgomet and Tanja Pascale Neuvians},
  journal={Biotechnology Letters},
  year={2004},
  volume={26},
  pages={509-515}
}
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 up- or 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… Expand
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References

SHOWING 1-10 OF 23 REFERENCES
Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes
TLDR
The normalization strategy presented here is a prerequisite for accurate RT-PCR expression profiling, which opens up the possibility of studying the biological relevance of small expression differences. Expand
Relative expression software tool (REST) for group-wise comparison and statistical analysis of relative expression results in real-time PCR.
TLDR
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. Expand
Housekeeping genes as internal standards: use and limits.
TLDR
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. Expand
Effect of experimental treatment on housekeeping gene expression: validation by real-time, quantitative RT-PCR.
TLDR
Beta-2 microglobulin and 18S rRNA are suitable internal control genes in quantitative serum-stimulation studies, while beta-actin and GAPDH are not. Expand
Semi-quantitative RT-PCR for comparison of mRNAs in cells with different amounts of housekeeping gene transcripts.
TLDR
This procedure enables transcripts to be compared when the differentiation process affects the transcription pattern of the beta-actin housekeeping gene, a commonly used internal standard, and is sensitive and avoids constructing internal competitive RNA standards. Expand
Regulation of hypoxanthine phosphoribosyltransferase, glyceraldehyde-3-phosphate dehydrogenase and beta-actin mRNA expression in porcine immune cells and tissues.
TLDR
Cloned from swine, three genes commonly used as endogenous controls in other species are cloned and their relative levels of expression in various porcine tissues and their response to various cell activators are characterized. Expand
Control selection for RNA quantitation.
TLDR
The uses and pitfalls of the most popular of these controls, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and beta-actin, are discussed, with special emphasis on precautions associated with the use of GAPDH. Expand
A new mathematical model for relative quantification in real-time RT-PCR.
  • M. Pfaffl
  • Biology, Medicine
  • Nucleic acids research
  • 2001
TLDR
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. Expand
Processing of gene expression data generated by quantitative real-time RT-PCR.
TLDR
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. Expand
Validities of mRNA quantification using recombinant RNA and recombinant DNA external calibration curves in real-time RT-PCR
TLDR
An Insulin-like growth factor-1 (IGF-1) RT-PCR in the LightCycler is developed, optimised and validated and concludes that external calibration curve using recDNA is a better model for the quantification of mRNA than the recRNA calibration model. Expand
...
1
2
3
...