Journal Article

Identification and Testing of Superior Reference Genes for a Starting Pool of Transcript Normalization in Arabidopsis

Sung Myun Hong, Sung Chul Bahn, Aram Lyu, Hye Seung Jung and Ji Hoon Ahn

in Plant and Cell Physiology

Published on behalf of Japanese Society of Plant Physiologists

Volume 51, issue 10, pages 1694-1706
Published in print October 2010 | ISSN: 0032-0781
Published online August 2010 | e-ISSN: 1471-9053 | DOI: http://dx.doi.org/10.1093/pcp/pcq128
Identification and Testing of Superior Reference Genes for a Starting Pool of Transcript Normalization in Arabidopsis

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  • Biochemistry
  • Molecular and Cell Biology
  • Plant Sciences and Forestry

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Genes that are stably expressed during development or in response to environmental changes are essential for accurate normalization in qRT–PCR experiments. To prevent possible misinterpretation caused by the use of unstable housekeeping genes, such as UBQ10, ACT, TUB and EF-1α, as a reference, the use of 20 stably expressed genes identified from microarray analyses was proposed. Furthermore, it was recommended that at least four genes among them be tested to identify suitable reference genes under different experimental conditions. However, testing the 20 potential reference genes under any condition is inefficient. Furthermore, since their stability still varies, there is a need to identify a subset of genes that are more stable than others, which can be used as a starting pool for testing. Here, we validated the expression stability of the potential candidate genes together with the above-mentioned conventional reference genes under six experimental conditions commonly used in plant developmental biology. To increase fidelity, three independent validation experiments were carried out for each experimental condition. A hypothetical normalization factor, which is the geometric mean of genes that were identified as stably expressed genes in each experiment, was used to exclude unstable genes under a given condition. We identified a subset of genes showing higher expression stability under specific experimental conditions. We recommend the use of these genes as a starting pool for the identification of suitable reference genes under given experimental conditions to ensure accurate normalization in qRT–PCR analysis.

Keywords: Normalization; qRT–PCR; Reference genes

Journal Article.  6433 words.  Illustrated.

Subjects: Biochemistry ; Molecular and Cell Biology ; Plant Sciences and Forestry

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