Selection of reference genes for quantitative real-time RT-PCR analysis in citrus

Abstract

Quantitative real-time reverse transcription polymerase chain reaction (qPCR) has become the preferred method for studying low-abundant mRNA expression. Appropriate application of qPCR in such studies requires the use of reference gene(s) as an internal control in order to normalize the mRNA levels between different samples for an exact comparison of gene expression levels. Expression of the reference gene should be independent from development stage, cell/tissue types, treatments and environmental conditions. Recognizing the importance of reference gene(s) in normalization of qPCR data, various reference genes have been evaluated for stable expression under specific conditions in various organisms. In plants, only a few of them have been investigated, and very few reports about such reference genes in citrus. In the present study, seven candidate reference genes (18SrRNA, ACTB, rpII, UBQI, UBQ10, GAPDH and TUB) were tested, and three of them (18SrRNA, ACTB and rpII) proved to be the most stable ones among six leaf samples of different citrus genotypes. The three candidate reference genes were further analyzed for their stability of expression in five different tissues, and the results indicated that they were not completely stable. It is commonly accepted that gene expression studies should be normalized using more than one reference gene. Based on our results, we propose the use of the mean result rendered by18SrRNA, ACTB and rpII as reference genes to normalize mRNA levels in qPCR analysis of diverse cultivars and tissues of citrus. These results may provide a guideline for future works on gene expression in citrus by using qPCR.

Selection of reference genes for expression analyses of red-fleshed sweet orange (Citrus sinensis)

“…In experiments with cacao, MDH also formed the best combination of reference genes with Rpl35 in tissues inoculated with M. perniciosa (Pinheiro et al, 2011). ACT has been evaluated as a gene reference in various gene expression studies (Liu et al, 2010;Chen et al, 2011), and was considered highly stable when analyzed in various Citrus genotypes (Yan et al, 2012). However, in the present study, ACT was one of the least stable genes, in both non-infected and infected plants.…”

“…The stability of genes that encode ribosomal proteins, such as Rpl35, has been evaluated in many studies, with positive results (Calsa and Figueira, 2007;Schmidt and Delaney, 2010;Pinheiro et al, 2011;Yan et al, 2012). The stability of seven reference gene candidates was analyzed in six Citrus genotypes by Yan et al (2012), and 18SrRNA was found to be one of the most stable.…”

“…The stability of seven reference gene candidates was analyzed in six Citrus genotypes by Yan et al (2012), and 18SrRNA was found to be one of the most stable. The expression stability of 10 candidates was evaluated in various tissues of Theobroma cacao by Pinheiro et al (2011), and Rpl35 was one of the most stable and suitable for the normalization of gene expression when outgrowing shoots were infected with the basidiomycete, Moniliophthora perniciosa.…”

“…The instability of reference gene expression may result in an over-or under-estimation of target gene expression . Gene expression studies in animals (Small et al, 2008) and plants, including Citrus species (Kou et al, 2012;Mafra et al, 2012;Yan et al, 2012), have demonstrated that even genes considered to have a constitutive expression exhibit variation in expression under different experimental conditions, which requires an a priori determination of the stability of reference gene candidates, depending upon the conditions to be evaluated.…”

Induced resistance in citrus by hexanoic acid

“…The primers used in the RT-qPCR were pth and PR2 previously described by , CALs1 described by and AOS and PR5 described by . Actin and 18S gene expression were used as an internal standard (Yan et al, 2012) (supplementary table 1: primer sequences).…”