Validation of Suitable Reference Genes for RT-qPCR Data in Achyranthes bidentata Blume under Different Experimental Conditions

Abstract

Real-time quantitative polymerase chain reaction (RT-qPCR) is a sensitive technique for gene expression studies. However, choosing the appropriate reference gene is essential to obtain reliable results for RT-qPCR assays. In the present work, the expression of eight candidate reference genes, EF1-α (elongation factor 1-α), GAPDH (glyceraldehyde 3-phosphate dehydrogenase), UBC (ubiquitin-conjugating enzyme), UBQ (polyubiquitin), ACT (actin), β-TUB (β-tubulin), APT1 (adenine phosphoribosyltransferase 1), and 18S rRNA (18S ribosomal RNA), was evaluated in Achyranthes bidentata samples using two algorithms, geNorm and NormFinder. The samples were classified into groups according to developmental stages, various tissues, stresses (cold, heat, drought, NaCl), and hormone treatments (MeJA, IBA, SA). Suitable combination of reference genes for RT-qPCR normalization should be applied according to different experimental conditions. In this study, EF1-α, UBC, and ACT genes were verified as the suitable reference genes across all tested samples. To validate the suitability of the reference genes, we evaluated the relative expression of CAS, which is a gene that may be involved in phytosterol synthesis. Our results provide the foundation for gene expression analysis in A. bidentata and other species of Amaranthaceae.

Keywords: Achyranthes bidentata Bl.; RT-qPCR data normalization; gene validation; reference genes; selection.

FIGURE 1

Specificity of primer pairs for RT-qPCR amplification. The 3% agarose gel electrophoresis showing a single product of the expected size for each candidate reference gene and the target gene CAS; M represents the DNA size marker.

FIGURE 2

Specificity of primer pairs for RT-qPCR amplification. Dissociation curves with single peaks generated for all amplicons.

FIGURE 3

RT-qPCR raw Cq values for candidate reference genes in different samples. The box-plot show the mean, maximum, minimum, interquartile range, and outliers.

FIGURE 4

Average expression stability values (M) of candidate reference genes. Average expression stability values (M) of the candidate reference genes were calculated by the geNorm software in A. bidentata samples under different experimental conditions, including Cold treatment (A), Heat treatment (B), development stages (Root) (C), development stages (Stem) (D), development stages (Leaf) (E), and different organs (F). The lowest M-value indicates the most stable gene and vice versa.

FIGURE 5

Average expression stability values (M) of the candidate reference genes. Average expression stability values (M) of the candidate reference genes were calculated by the geNorm software in A. bidentata samples under different experimental conditions, including IBA treatment (A), MeJA treatment (B), NaCl treatment (C), Drought treatment (D), SA treatment (E), and Total (F). The lowest M-value indicates the most stable gene and vice versa.

FIGURE 6

Pairwise variation (V) analysis of candidate reference genes in the A. bidentata sample sets. The Pairwise variation (Vn/Vn+1) between the normalization factors was calculated using the geNorm software program to determine the optimal number of candidate reference genes.

FIGURE 7

Relative expression levels of CAS using validated reference genes for normalization under the heat stress treatment. The validated reference gene (s) used as normalization factors were alone (ACT and EF1-α) or the combination (ACT and EF1-α) of most stable reference genes, and the most unsuitable one (18S rRNA) in heat stress sample set. The expression level of pre-treatment samples (0 h) was set to a value of 1. Each value represents the mean of three replicates, standard error bars are shown.