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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Careful selection of reference genes is required for reliable performance of RT-qPCR in human normal and cancer cell lines
Francis Jacob,Francis Jacob,Rea Guertler,Stephanie Naim,Sheri Nixdorf,André Fedier,Neville F. Hacker,Viola Heinzelmann-Schwarz,Viola Heinzelmann-Schwarz,Viola Heinzelmann-Schwarz +9 more
TL;DR: The aim was to review the literature since implementation of the MIQE guidelines in order to identify the degree of acceptance; compare various algorithms in their expression stability; and identify a set of suitable and most reliable reference genes for a variety of human cancer cell lines.
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Gene expression changes related to the production of phenolic compounds in potato tubers grown under drought stress.
Christelle M. Andre,Roland Schafleitner,Sylvain Legay,Isabelle S. Lefèvre,Carlos Alberto Alvarado Aliaga,Giannina Nomberto,Lucien Hoffmann,Jean-Francois Hausman,Yvan Larondelle,Danièle Evers +9 more
TL;DR: It is proposed that the altered sucrose flux induced by the drought stress is partly responsible for the changes in gene expression, which could be useful in the development of potato varieties with enhanced health and nutritional benefits.
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Gene amplification as double minutes or homogeneously staining regions in solid tumors: Origin and structure
Clelia Tiziana Storlazzi,Angelo Lonoce,M. C. Guastadisegni,Domenico Trombetta,Pietro D'Addabbo,Giulia Daniele,Alberto L'Abbate,Gemma Macchia,Cecilia Surace,Klaas Kok,Reinhard Ullmann,Stefania Purgato,Orazio Palumbo,Massimo Carella,Peter F. Ambros,Mariano Rocchi +15 more
TL;DR: This paper investigates 10 cell lines from solid tumors showing MYCN amplification as dmin or hsr, and strongly argues that the episome model applies to solid tumors as well, and that dmin and hsr are two faces of the same coin.
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ABCA Transporter Gene Expression and Poor Outcome in Epithelial Ovarian Cancer
Ellen L. Hedditch,Bo Gao,Amanda J. Russell,Yi Lu,Catherine Emmanuel,Jonathan Beesley,Sharon E. Johnatty,Xiaoqing Chen,Paul R. Harnett,Joshy George,Rebekka T. Williams,Claudia Flemming,Diether Lambrechts,Evelyn Despierre,Sandrina Lambrechts,Ignace Vergote,Beth Y. Karlan,Jenny Lester,Sandra Orsulic,Christine Walsh,Peter A. Fasching,Peter A. Fasching,Matthias W. Beckmann,Arif B. Ekici,Alexander Hein,Keitaro Matsuo,Satoyo Hosono,Toru Nakanishi,Yasushi Yatabe,Tanja Pejovic,Yukie Bean,Florian Heitz,Florian Heitz,Philipp Harter,Andreas du Bois,Ira Schwaab,Estrid Høgdall,S.K. Kjaer,Allan Jensen,Claus Høgdall,Lene Lundvall,Svend Aage Engelholm,Bob Brown,James M. Flanagan,Michelle D Metcalf,Nadeem Siddiqui,Thomas A. Sellers,Brooke L. Fridley,Julie M. Cunningham,Joellen M. Schildkraut,Ed Iversen,Rachel Palmieri Weber,Andrew Berchuck,Ellen L. Goode,David D.L. Bowtell,Georgia Chenevix-Trench,Anna deFazio,Murray D. Norris,Stuart MacGregor,Michelle Haber,Michelle J. Henderson +60 more
TL;DR: In this article, the relationship between clinical outcomes and ABC transporter gene expression in two independent cohorts of high-grade serous EOC tumors was assessed with real-time quantitative polymerase chain reaction, analysis of expression microarray data, and immunohistochemistry.
Journal ArticleDOI
Impact of normalization on miRNA microarray expression profiling.
Sylvain Pradervand,Johann Weber,Jérôme Thomas,Manuel Bueno,Pratyaksha Wirapati,Karine Lefort,G. Paolo Dotto,Keith Harshman +7 more
TL;DR: A method to select nonchanging miRNAs (invariants) and use them to compute linear regression normalization coefficients or variance stabilizing normalization (VSN) parameters is developed and can be applied to other data sets including those from one color miRNA microarray platforms, focused gene expression arrays, and gene expression analysis using quantitative PCR.
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