Assessment of reference gene stability in Rice stripe virus and Rice black streaked dwarf virus infection rice by quantitative Real-time PCR.

Linlin Du,Yijun Zhou,Tong Zhou,Ying Lan,Peng Fang,Rongfei Lu,Wenbiao Shen,Feng Sun

doi:10.1186/s12985-015-0405-2

Abstract

BackgroundStably expressed reference gene(s) normalization is important for the understanding of gene expression patterns by quantitative Real-time PCR (RT-qPCR), particularly for Rice stripe virus (RSV) and Rice black streaked dwarf virus (RBSDV) that caused seriously damage on rice plants in China and Southeast Asia.MethodsThe expression of fourteen common used reference genes of Oryza sativa L. were evaluated by RT-qPCR in RSV and RBSDV infected rice plants. Suitable normalization reference gene(s) were identified by geNorm and NormFinder algorithms.ResultsUBQ 10 + GAPDH and UBC + Actin1 were identified as suitable reference genes for RT-qPCR normalization under RSV and RBSDV infection, respectively. When using multiple reference genes, the expression patterns of OsPRIb and OsWRKY, two virus resistance genes, were approximately similar with that reported previously. Comparatively, by using single reference gene (TIP41-Like), a weaker inducible response was observed.ConclusionsWe proposed that the combination of two reference genes could obtain more accurate and reliable normalization of RT-qPCR results in RSV- and RBSDV-infected plants. This work therefore sheds light on establishing a standardized RT-qPCR procedure in RSV- and RBSDV-infected rice plants, and might serve as an important point for discovering complex regulatory networks and identifying genes relevant to biological processes or implicated in virus.Electronic supplementary materialThe online version of this article (doi:10.1186/s12985-015-0405-2) contains supplementary material, which is available to authorized users.

Highlights

Expressed reference gene(s) normalization is important for the understanding of gene expression patterns by quantitative Real-time PCR (RT-qPCR), for Rice stripe virus (RSV) and Rice black streaked dwarf virus (RBSDV) that caused seriously damage on rice plants in China and Southeast Asia
Our results further indicated that the combination of these two reference genes provides a good starting point for gene expression analysis in rice viral infection plants by RT-qPCR
Rice infectivity assay We characterized the phenotype of RSV- and RBSDVinfected rice plants, and the symptoms were allowed to develop under the controlled environmental conditions

Summary

Introduction

Expressed reference gene(s) normalization is important for the understanding of gene expression patterns by quantitative Real-time PCR (RT-qPCR), for Rice stripe virus (RSV) and Rice black streaked dwarf virus (RBSDV) that caused seriously damage on rice plants in China and Southeast Asia. The reference genes, such as those encoding actin (Actin), tubulin (TUB), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and 18S rRNA, are often separately chosen for the normalization in RT-qPCR because of their constant expression levels in living organisms [16]. It is important and necessary to select suitable reference gene(s) for different experimental paradigms, in RSV and RBSDV infection conditions which those appropriate internal reference(s) were not identified [25,26,27]

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Conclusion