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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Overexpression of Arabidopsis ECERIFERUM1 Promotes Wax Very-Long-Chain Alkane Biosynthesis and Influences Plant Response to Biotic and Abiotic Stresses
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TL;DR: The characterization of the Arabidopsis ECERIFERUM1 (CER1) gene predicted to encode an enzyme involved in alkane biosynthesis demonstrates that CER1 controls alkanes biosynthesis and is highly linked to responses to biotic and abiotic stresses.
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Identification and evaluation of new reference genes in Gossypium hirsutum for accurate normalization of real-time quantitative RT-PCR data
Sinara Artico,Sarah Muniz Nardeli,Osmundo Brilhante,Maria Fatima Grossi-de-Sa,Marcio Alves-Ferreira +4 more
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Chemerin activation by serine proteases of the coagulation, fibrinolytic, and inflammatory cascades.
Brian A. Zabel,Brian A. Zabel,Samantha J. Allen,Paulina Kulig,Jessica A. Allen,Jessica A. Allen,Joanna Cichy,Tracy M. Handel,Eugene C. Butcher,Eugene C. Butcher +9 more
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The obesity gene, FTO, is of ancient origin, up-regulated during food deprivation and expressed in neurons of feeding-related nuclei of the brain
Robert Fredriksson,Maria Hägglund,Pawel K. Olszewski,Olga Stephansson,Josefin A. Jacobsson,Agnieszka M. Olszewska,Allen S. Levine,Jonas Lindblom,Helgi B. Schiöth +8 more
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Identification of suitable internal control genes for expression studies in Coffea arabica under different experimental conditions
TL;DR: Gapdh is the recommended reference gene for measuring gene expression in Coffea arabica and will enable more accurate and reliable normalization of tissue/organ-specific gene expression studies in this important cherry crop plant.
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