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Measuring ‘normalcy’ in plant gene expression after herbivore attack

Plants make drastic changes to their transcriptome to appropriately respond to environmental change, and the regulation of genes that are specific to abiotic and biotic stresses is a key to plant survival. The coordination of defence gene transcription is often coupled with significant adjustments in the levels of expression of primary metabolic and structural genes to relocate resources, repair damage and/or induce senescence. This complicates the process of finding suitable ‘housekeeping’ or reference genes to use in measurements of gene expression by real‐time reverse transcription (RT–PCR) in response to herbivore attack. Several software programs have been developed to identify candidate reference genes, but measurement of their expression may still not yield an appropriate gene or suite of genes for normalization. This is especially true in plant–herbivore interactions where tissue damage is immediate and continuous. Here, we show that 12 traditional reference genes customarily used in RT–PCR analysis are not stably expressed after insect attack. We describe the pitfalls of using traditional reference genes and why insect attack may be affecting whole cell metabolism. We propose a method using RNA quantification in combination with an external spike of commercially available mRNA as normalization factors in studies involving herbivory, multiple stress treatments or species where stable reference genes are unknown.