Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes
Jo Vandesompele,Katleen De Preter,Filip Pattyn,Bruce Poppe,Nadine Van Roy,Anne De Paepe,Franki Speleman +6 more
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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.Abstract:
Gene-expression analysis is increasingly important in biological research, with real-time reverse transcription PCR (RT-PCR) becoming the method of choice for high-throughput and accurate expression profiling of selected genes. Given the increased sensitivity, reproducibility and large dynamic range of this methodology, the requirements for a proper internal control gene for normalization have become increasingly stringent. Although housekeeping gene expression has been reported to vary considerably, no systematic survey has properly determined the errors related to the common practice of using only one control gene, nor presented an adequate way of working around this problem. We outline a robust and innovative strategy to identify the most stably expressed control genes in a given set of tissues, and to determine the minimum number of genes required to calculate a reliable normalization factor. We have evaluated ten housekeeping genes from different abundance and functional classes in various human tissues, and demonstrated that the conventional use of a single gene for normalization leads to relatively large errors in a significant proportion of samples tested. The geometric mean of multiple carefully selected housekeeping genes was validated as an accurate normalization factor by analyzing publicly available microarray data. The normalization strategy presented here is a prerequisite for accurate RT-PCR expression profiling, which, among other things, opens up the possibility of studying the biological relevance of small expression differences.read more
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Comprehensive selection of reference genes for gene expression normalization in sugarcane by real time quantitative rt-PCR.
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Assessing the Effects of Light on Differentiation and Virulence of the Plant Pathogen Botrytis cinerea: Characterization of the White Collar Complex
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Response to soy: T‐cell‐like reactivity in the intestine of Atlantic salmon, Salmo salar L.
A.M. Bakke-McKellep,Marianne K. Frøystad,Einar Lilleeng,F Dapra,Ståle Refstie,Åshild Krogdahl,Thor Landsverk +6 more
TL;DR: A T-cell-like response appears to be involved in this example of a food-sensitive enteropathy in salmon.
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Reference gene selection for quantitative real-time PCR in Chrysanthemum subjected to biotic and abiotic stress.
TL;DR: The catalytic subunit of protein phosphatase 2A was the best performing one during heat and waterlogging stress, but was the worst during aphid infestation; the commonly used reference gene actin was generally the least stable of the set.
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Hypothalamic FTO is associated with the regulation of energy intake not feeding reward
Pawel K. Olszewski,Robert Fredriksson,Agnieszka M. Olszewska,Olga Stephansson,Johan Alsiö,Katarzyna J. Radomska,Allen S. Levine,Helgi B. Schiöth +7 more
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