Selection of reliable reference genes for quantitative real-time polymerase chain reaction studies in maize grains

Abstract

The stability of candidate reference genes was evaluated in maize landrace varieties and during multiple grain developmental stages to evaluate the expression of carotenoid-related genes by RT-qPCR for application to maize biofortification. Vitamin A deficiency affects millions of children worldwide; therefore, increasing the content of vitamin A precursors in maize grains is of interest. The study of the expression of genes involved in the carotenoid biosynthetic pathway in maize grains has provided useful information for metabolic engineering approaches. However, reliable results using real-time quantitative polymerase chain reaction (RT-qPCR) experiments are dependent on the use of the appropriate reference genes. In this study, we utilized geNorm and NormFinder softwares to identify the most stably expressed candidate reference genes in samples from seven stages of grain development and from eight landrace varieties. The results of the analysis performed using geNorm indicated that tubulin (TUB) and actin (ACT) were the most suitable reference genes among all experimental conditions, while glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH) showed the least stability. The same result was obtained with the NormFinder software. The minimum number of genes required in each experimental condition to normalize the gene expression data was also determined by geNorm. The expression of phytoene synthase gene (PSY1), the first enzyme in the carotenoid biosynthetic pathway, was overestimated when the least stable candidate gene (GAPDH) was used as the internal control instead of the most stable gene pair (ACT + TUB), thus highlighting the importance of validating reference genes before conducting a RT-qPCR experiment to obtain accurate results. This study is the first survey of the stability of genes for use as reference genes to normalize RT-qPCR data from maize landraces during multiple stages of grain development.

Genome-wide identification of housekeeping genes in maize

“…The rest of the reference gene candidates were also protein coding genes, including RPN, UPF1 and SGT1. Although GAPDH has been widely used as an internal control including studies in maize (Murphy et al 2010), it showed the least stability in qPCR analysis, which echoed the conclusions from Galli et al (2013).…”

“…In maize, several traditional housekeeping genes were used widely as internal controls, including ACT (Danilevskaya et al 2008), TUB (Ding et al 2009), MEZ1 (Makarevitch et al 2008), GAPDH (Murphy et al 2010), etc. Galli et al (2013) considered TUB and ACT as the most suitable reference genes among five candidates (18S, ACT, GAPDH, TUB, and UBI) in seven stages of maize grain development. However, none of these traditional reference genes were included in the 48 and 589 genes in this study (Supplementary Table S1; Supplementary Table S2), and the qPCR result indicated that ACT and GAPDH are more variable compared with our reference gene candidates under nitrogen treatments (Fig.…”

“…In maize, which is one of the most important crops all over the world with rich genetic and phenotypic diversity, RNAseq data has also been used, such as in reproductive transcriptomes analysis (Davidson et al 2011). Several studies analyzed a few candidate reference genes in maize Manoli et al 2012;Galli et al 2013). But to date, no systematic research on housekeeping genes in maize has been reported.…”

“…Remans et al (2008) identified the best three reference genes from a set of ten candidates in Arabidopsis for normalization of gene expression. In maize, Galli et al (2013) examined five candidate reference genes and declared TUB and ACT were the most suitable reference genes among samples from seven stages of maize grain development. Manoli et al (2012) analyzed 12 genes in a set of 20 samples and found several new genes including CUL, FPGS, LUG, MEP and UBCP which exhibited the highest expression stability other than the current widely used reference genes.…”

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

“…The objective of this study was to evaluate the stability of 11 candidate genes using various tissue and organ samples from healthy plants or leaves from citrus greening disease (Huanglongbing)-symptomatic plants of a Brazilian red-fleshed cultivar ('Sanguínea de Mombuca'), in order to select the most suitable reference gene for investigating gene expression under these conditions. geNorm and NormFinder identified genes that encoded translation initiation Usually, reference genes encode for proteins with a constitutive expression, such as actin (ACT) (Liu et al, 2010), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Galli et al, 2013), elongation factors (EF) (Bomal et al, 2008), ubiquitin (UBQ) (Lin and Lai, 2010), and tubulin (TUB) (Die et al, 2010), which are all part of the basic metabolism of plant cells, and therefore are assumed to exhibit little variation in expression under different ontogenetic or biological conditions. The instability of reference gene expression may result in an over-or under-estimation of target gene expression .…”