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

Nancy Podevin,Rony Swennen,Serge Remy,An Krauss,Isabelle Henry

doi:10.1007/s11032-012-9711-1

Abstract

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

Highlights

Reverse transcription quantitative polymerase chain reaction (RT-qPCR) is a routinely used technique for gene expression analysis because of its main advantages of relatively low cost, good speed, a wide dynamic range, and feasibility in non-model organisms (Thellin et al 1999)
Extreme care needs to be exercised in the interpretation of real-time or quantitative polymerase chain reaction (RT-qPCR) data and, in particular, normalization is crucial to control for experimental errors that can be introduced at a number of stages throughout the procedure
Reference genes commonly used for other plant species were investigated to identify genes displaying highly uniform expression patterns in different varieties, tissues, developmental stages, and stress conditions for the non-model crop Musa

Summary

Introduction

Reverse transcription quantitative polymerase chain reaction (RT-qPCR) is a routinely used technique for gene expression analysis because of its main advantages of relatively low cost, good speed, a wide dynamic range, and feasibility in non-model organisms (Thellin et al 1999). The most reliable method of normalization involves the use of one or preferably more housekeeping or reference genes as internal standards. The expression of these reference genes is expected to remain constant under different experimental conditions. Used reference genes are cellular maintenance genes, which regulate basic and ubiquitous cellular functions such as components of the cytoskeleton, glycolytic pathway, protein folding, synthesis of ribosome subunits, electron transport, and protein degradation (Gachon et al 2004; Huggett et al 2005). Recent studies have shown that the transcriptional levels of these reference genes are not always stable, and that no single reference gene has a constant expression level under all experimental

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