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Identification and Validation of Reference Genes for Normalization of Transcripts from Virus-Infected Arabidopsis thaliana

March 2011 , Volume 24 , Number  3
Pages  294 - 304

S. T. Lilly,1,2 R. S. M. Drummond,1 M. N. Pearson,2 and R. M. MacDiarmid1

1The New Zealand Institute for Plant and Food Research Limited. Auckland, New Zealand; 2School of Biological Sciences, University of Auckland, Auckland, New Zealand

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Accepted 12 November 2010.

Real-time quantitative polymerase chain reaction (qPCR) of complementary DNA is now a standard method for studies of gene expression. However, qPCR can identify genuine variation only when transcript quantities are accurately normalized to an appropriate reference. To identify the most reliable reference genes for transcript quantification by qPCR, we describe a systematic evaluation of candidate reference genes of Arabidopsis thaliana ecotype Columbia-0 (Col-0). Twelve genes were selected for transcript stability studies by qPCR of complementary DNA prepared from Arabidopsis leaf tissue infected with one of five plant viruses (Cauliflower mosaic virus, Tobacco mosaic virus, Tomato spotted wilt virus, Turnip mosaic virus, and Turnip yellow mosaic virus). The F-box family protein, elongation factor 1-α, sand family protein, and protodermal factor 2 gene transcripts showed the most stable accumulation, whereas a traditionally used reference gene, Actin8, showed the least stable accumulation as measured by the geNorm algorithm. The data furnish plant virologists with reference genes for normalization of qPCR-derived gene expression in virus-infected Arabidopsis and will be beneficial to the selection and design of primers targeting orthologous genes in other plant species.

© 2011 The American Phytopathological Society