Reference Gene Selection for Quantitative Real-time PCR Analyses of Acer palmatum under Abiotic Stress
Abstract. Quantitative real-time reverse transcriptase PCR (qRT-PCR) technology has been extensively used to estimate gene expression levels, and the selection of appropriate reference genes for qRT-PCR analysis is critically important for obtaining authentic normalized data. Acer palmatum is an important colorful leaf ornamental tree species, and reference genes suitable for normalization of the qRT-PCR data obtained from this species have not been investigated. In this study, the expression stability of ten candidate reference genes, namely, Actin3, Actin6, Actin9, EF1α, PP2A, SAMDC, TIP41, TUBα, TUBβ and UBQ10, in two distinct tissues (leaves and roots) of A. palmatum under four different abiotic (cold, heat, salt and drought) stress conditions were investigated and assessed using three statistical methods (GeNorm, NormFinder and BestKeeper). The combinations of reference genes that showed stability in the different stressed samples were different. Specifically, Actin3, Actin6, Actin9 and UBQ10 were the most stable reference genes in all the samples, and Actin3 and Actin6 were stably expressed in cold-stressed leaves and roots. Actin3, Actin6 and UBQ10 were identified as an appropriate combination of reference genes for the analysis of heat-stressed leaves and roots, whereas the combination of Actin9, UBQ10 and Actin6 was deemed the most suitable for the analysis of salt-stressed leaves and roots. Similarly, Actin6 and UBQ10 exhibited stable expression in drought-stressed leaves and roots. Furthermore, the expression levels of ApCBF were estimated to determine the reliability of the reference genes assessed in this study. This study revealed stable reference genes in A. palmatum that might be used for the normalization of qRT-PCR data obtained under various abiotic stresses.
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