Reference Gene Selection for Quantitative Real-Time PCR in Chrysanthemum Subjected to Biotic and Abiotic Stress

Abstract

Quantitative real-time PCR (RT-qPCR) is a reliable method for assessing gene expression, provided that suitable reference genes are included to normalize the data. The stability of expression of eight potential reference genes, namely, tubulin (alpha-2,4 tubulin), actin, EF1 α (elongation factor 1 α), UBC (ubiquitin C), GAPDH (glyceraldehyde-3-phosphate dehydrogenase), psaA (photosynthesis-related plastid gene representing photosystem I), PP2Acs (catalytic subunit of protein phosphatase 2A), and PGK (phosphoglycerate kinase), was assessed in chrysanthemum plants subjected to aphid infestation, heat stress or waterlogging stress using geNorm software. The widely used reference gene EF1 α performed well for aphid infested plants but poorly for waterlogged ones. The catalytic subunit of protein phosphatase 2A (PP2Acs) was the best performing one during heat and waterlogging stress, but was the worst during aphid infestation. The commonly used reference gene actin was generally the least stable of the set. No single gene was suitable for normalization on its own. The choice of reference gene(s) is an important factor in gene expression studies based on RT-qPCR.

Reference gene selection in Artemisia annua L., a plant species producing anti-malarial artemisinin

“…In the present study, the EF1a gene appeared to be stably expressed over a wide range of experimental conditions. Under temperature treatment, EF1a was the most stably expressed gene in A. annua, and the results were consistent with the previously reported results from chrysanthemum, a plant species belonging to the family of Compositae (Gu et al 2011). EF1a was also recommended to be the suitable reference gene under the treatment of plant growth regulators in zucchini (Obrero et al 2011) and Catharanthus roseus (Wei 2010).…”

Reference genes assessment for the seagrass Posidonia oceanica in different salinity, pH and light conditions

“…Furthermore, in comparing the NormFinder and geNorm approaches, Andersen et al (2004) showed that NormFinder may be more sensible than geNorm in detecting unstable gene expression (see also Ransbotyn and Reusch 2006) and recommended using the best pair of RGs given by NormFinder. This is why we also analyzed the GOI expression levels using the two best RGs by NormFinder to normalize the RT-qPCR, although also geNorm alone has been used for suitable RG assessment in other studies (Nicot et al 2005;Lovdal and Lillo 2009;Gu et al 2011). Giving the complementarity in the information obtained with the three different software, and the lack in the literature of a general consensus on the best approach to use, we suggest to use all of the three software in order to have a more accurate RG assessment.…”

Reference gene selection for gene expression studies in lily using quantitative real-time PCR

“…There have been many reports on reference gene validation in various plants, such as rice (Jain et al, 2006), chickpea (Garg et al, 2010), soybean (Jian et al, 2008;Hu et al, 2009), tomato (Expósito-Rodríguez et al, 2008), chrysanthemum (Gu et al, 2011), and poplar (Gutierrez et al, 2008). The results illustrate that candidate reference genes may vary among species and even among different cultivars.…”