Identification and Validation of Reference Genes for qRT-PCR Studies of Gene Expression in Dioscorea opposita

Abstract

Quantitative real-time polymerase chain reaction (qRT-PCR) is one of the most common methods for gene expression studies. Data normalization based on reference genes is essential for obtaining reliable results for qRT-PCR assays. This study evaluated potential reference genes of Chinese yam (Dioscorea opposita Thunb.), which is an important tuber crop and medicinal plant in East Asia. The expression of ten candidate reference genes across 20 samples from different organs and development stages was assessed. We identified the most stable genes for qRT-PCR studies using combined samples from different organs. Our results also suggest that different suitable reference genes or combinations of reference genes for normalization should be applied according to different organs and developmental stages. To validate the suitability of the reference genes, we evaluated the relative expression of PE2.1 and PE53, which are two genes that may be associated with microtuber formation. Our results provide the foundation for reference gene(s) selection in D. opposita and will contribute toward more accurate gene analysis studies of the genus Dioscorea.

Figure 1

Specificity of qRT-PCR amplicons. (a) Agarose gel electrophoresis showing amplification of a single product of the expected size for each candidate gene and two target genes for reference gene validation. M represents DL2,000 DNA marker. (b) Dissociation curves with single peaks generated for all amplicons.

Figure 2

RNA transcription levels of reference genes tested, presented as Ct mean value in the different samples. Boxes indicate the 25th/75th percentiles, lines across the boxes depict the medians, squares represent the means, and whiskers indicate the ranges for all samples.

Figure 3

Gene expression stability and rankings of ten candidate reference genes, as calculated by geNorm software. The average expression stability (M) was calculated following stepwise exclusion of the least stable gene across all the samples within an experimental set. The lowest M value indicates the most stable gene, while the highest value represents the most highly variable gene.

Figure 4

Pairwise variation (V) analysis of the candidate reference genes. The pairwise variation (Vn/n + 1) was analyzed between two sequential normalization factors NFn and NFn + 1 that contained an increasing number of reference genes using geNorm software. Vn/n + 1 < 0.15 indicates that the inclusion of an additional reference gene is not required.

Figure 5

Relative expression levels of PE2.1 (a) and PE53 (b) during three different development stages of microtuber formation, normalized by different combinations of reference genes, as indicated. APT was found to be one of the most stable genes by both geNorm and NormFinder. The combination of TUB and UBQ was the optimal combination as selected by geNorm. The combination of APT and ACT was found to be optimal in the NormFinder analysis. The four most stable reference genes were suggested by both analyses. Standard error bars are indicated.