Selection and validation of reference genes for quantitative RT-PCR expression studies of the non-model crop MusaReport as inadecuate

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Molecular Breeding

, Volume 30, Issue 3, pp 1237–1252

First Online: 08 June 2012Received: 21 February 2011Accepted: 08 February 2012


Gene expression analysis by reverse transcriptase real-time or quantitative polymerase chain reaction RT-qPCR is becoming widely used for non-model plant species. Given the high sensitivity of this method, normalization using multiple housekeeping or reference genes is critical, and careful selection of these reference genes is one of the most important steps to obtain reliable results. In this study, reference genes commonly used for other plant species were investigated to identify genes displaying highly uniform expression patterns in different varieties, tissues, developmental stages, fungal infection, and osmotic stress conditions for the non-model crop Musa banana and plantains. The expression stability of six candidate reference genes was tested on six different sample sets, and the results were analyzed using the publicly available algorithms geNorm and NormFinder. Our results show that variety, plant material, primer set, and gene identity can all influence the robustness and outcome of RT-qPCR analysis. In the case of Musa, a combination of three reference genes EF1, TUB and ACT can be used for normalization of gene expression data from greenhouse leaf samples. In the case of shoot meristem cultures, numerous combinations can be used because the investigated reference genes exhibited limited variability. In contrast, variability in expression of the reference genes was much larger among leaf samples from plants grown in vitro, for which the best combination of reference genes L2 and ACT genes is still suboptimal. Overall, our data confirm that the stability of candidate reference genes should be thoroughly investigated for each experimental condition under investigation.

KeywordsMusa Banana Reference genes qPCR Gene expression Mycosphaerella Nancy Podevin and An Krauss have contributed equally to this work.

Nancy Podevin: The views and positions expressed in this work are solely those of the author, who acts in his personal capacity, and do not necessarily represent the views of the EFSA or of the European Union.

Electronic supplementary materialThe online version of this article doi:10.1007-s11032-012-9711-1 contains supplementary material, which is available to authorized users.

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Author: Nancy Podevin - An Krauss - Isabelle Henry - Rony Swennen - Serge Remy


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