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Selection and validation of reference genes for quantitative RT-PCR expression studies of the non-model crop Musa.
MedLine Citation:
PMID:  23024595     Owner:  NLM     Status:  Publisher    
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. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11032-012-9711-1) contains supplementary material, which is available to authorized users.
Nancy Podevin; An Krauss; Isabelle Henry; Rony Swennen; Serge Remy
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Publication Detail:
Type:  JOURNAL ARTICLE     Date:  2012-6-8
Journal Detail:
Title:  Molecular breeding : new strategies in plant improvement     Volume:  30     ISSN:  1380-3743     ISO Abbreviation:  Mol. Breed.     Publication Date:  2012 Oct 
Date Detail:
Created Date:  2012-10-1     Completed Date:  -     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  9506703     Medline TA:  Mol Breed     Country:  -    
Other Details:
Languages:  ENG     Pagination:  1237-1252     Citation Subset:  -    
Laboratory of Tropical Crop Improvement, Department of Biosystems, Faculty of Bioscience Engineering, K.U. Leuven, Kasteelpark Arenberg 13, 3001 Leuven, Belgium ; European Food Safety Authority (EFSA), Largo N. Palli 5/A, 43121 Parma, Italy.
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