Selection of Reliable Reference Genes for Gene Expression Studies on Rhododendron molle G. Don

Abstract

The quantitative real-time polymerase chain reaction (qRT-PCR) approach has become a widely used method to analyze expression patterns of target genes. The selection of an optimal reference gene is a prerequisite for the accurate normalization of gene expression in qRT-PCR. The present study constitutes the first systematic evaluation of potential reference genes in Rhododendron molle G. Don. Eleven candidate reference genes in different tissues and flowers at different developmental stages of R. molle were assessed using the following three software packages: GeNorm, NormFinder, and BestKeeper. The results showed that EF1-α (elongation factor 1-alpha), 18S (18s ribosomal RNA), and RPL3 (ribosomal protein L3) were the most stable reference genes in developing rhododendron flowers and, thus, in all of the tested samples, while tublin (TUB) was the least stable. ACT5 (actin), RPL3, 18S, and EF1-α were found to be the top four choices for different tissues, whereas TUB was not found to favor qRT-PCR normalization in these tissues. Three stable reference genes are recommended for the normalization of qRT-PCR data in R. molle. Furthermore, the expression profiles of RmPSY (phytoene synthase) and RmPDS (phytoene dehydrogenase) were assessed using EF1-α, 18S, ACT5, RPL3, and their combination as internals. Similar trends were found, but these trends varied when the least stable reference gene TUB was used. The results further prove that it is necessary to validate the stability of reference genes prior to their use for normalization under different experimental conditions. This study provides useful information for reliable qRT-PCR data normalization in gene studies of R. molle.

Selection of suitable reference genes for qRT-PCR studies during SE initial dedifferentiation in cotton of different SE capability

“…However, other studies indicated that the analytical results obtained using NormFinder were consistent with those obtained with GeNorm, for example, in the halophyte Halostachys caspica , Chrysanthemum morifolium and Chrysanthemum lavandulifolium , rice and Rhododendron molle G. Don 30 . In this study, the ranking of NormFinder was relatively consistent with analysis of GeNorm.…”

“…The divergence ranking may have been due to the different algorithms 31 . Most studies of Bestkeeper have utilized CV and SD of the Cq to evaluate the stability and expression of reference genes 30,32 . Besides, as for BestKeeper, which is based on the R of the BestKeeper index by calculating the SD and CV 30,33,34 .…”

Transcriptome-based validation of proper reference genes for reverse trascription quantitative PCR analysis of Sinocalycanthus chinensis

“…However, the last ranking order by the two methods was identical in all sample sets. Similarly, based on the rank, the most unstable gene was also the same in many other plant species when different statistical algorithms were used , Zhang et al 2015, Xiao et al 2016). In addition, the two most stable genes generated by the two methods were identical in most sample sets.…”

“…Relative quantification of ScANS expression in outer and inner petals at different developmental stages using different reference genes (ScEF, ScPP2A, ScRPL, and ScPGK) and a combination of reference genes (ScEF+ScPP2A). Means ± SDs, n = 3. gene are often found to serve as suitable reference genes in other plant species, such as wheat (Liu et al 2014), tomato (Løvdal and Lillo 2009), Theobroma cacao (Pinheiro et al 2011), and Rhododendron molle (Xiao et al 2016). In developing flowers and different tissues of R. molle, the RPL gene was recommended for the normalization of RT-qPCR data (Xiao et al 2016).…”

“…However, the expression performance of reference genes is quite different in different plant species or under different experimental conditions. For instance, 18S is considered a dependable reference gene in developing Rhododendron molle flowers (Xiao et al 2016), but this gene is not recommended as a suitable reference gene in Osmanthus fragrans (Zhang et al 2015).…”