Selection and Validation of Reference Genes for mRNA Expression by Quantitative Real-Time PCR Analysis in Neolamarckia cadamba

Abstract

Neolamarckia cadamba is an economically-important fast-growing tree species in South China and Southeast Asia. As a prerequisite first step for future gene expression studies, we have identified and characterized a series of stable reference genes that can be used as controls for quantitative real time PCR (qRT-PCR) expression analysis in this study. The expression stability of 15 candidate reference genes in various tissues and mature leaves under different conditions was evaluated using four different algorithms, i.e., geNorm, NormFinder, BestKeeper and RefFinder. Our results showed that SAMDC was the most stable of the selected reference genes across the set of all samples, mature leaves at different photosynthetic cycles and under drought stress, whereas RPL10A had the most stable expression in various tissues. PGK and RPS25 were considered the most suitable reference for mature leaves at different developmental stages and under cold treatment, respectively. Additionally, the gene expression profiles of sucrose transporter 4 (NcSUT4), and 9-cis-epoxycarotenoid dioxygenase 3 (NcNCED3) were used to confirm the validity of candidate reference genes. Collectively, our study is the first report to validate the optimal reference genes for normalization under various conditions in N. cadamba and will benefit the future discovery of gene function in this species.

Figure 1

Distribution of threshold cycle (CT) values of 15 candidate reference genes across all 22 samples. The solid line within each box represents the 50th percentile. The lower boundary and upper boundary of each box represents the 25th and 75th percentile, respectively. The circles represent potential outliers.

Figure 2

Average expression stability values (M) of 15 candidate reference genes calculated by geNorm. (a) Tissue; (b) developmental stage; (c) photosynthetic cycles; (d) drought treatment; (e) cold treatment; (f) all of the samples in our given conditions.

Figure 3

Determination of the optimal number of reference genes for normalization in the tested experimental conditions. geNorm was used to calculate the normalization factor (NF) from at least two genes; the variable V defines the pair-wise variation between two sequential NF values.

Figure 4

The stability value of 15 candidate reference genes calculated by NormFinder. The lower value indicated the higher stability of gene expression. (a) Tissue; (b) developmental stage; (c) photosynthetic cycles; (d) drought treatment; (e) cold treatment; (f) all of the samples in our given conditions.

Figure 5

Relative quantification of NcSUT4 and NcNCED3 expression levels using the most and least stable reference genes for normalization in the given experimental conditions. (a) Expression level of NcSUT4 at different developmental stages. Most stable reference genes (SAMDC, PGK) and least stable reference genes (ACT11, EF-1-α) were used, respectively; (b) expression level of NcSUT4 in different tissues. Most stable reference genes (SAMDC, RPL10A) and least stable reference genes (GAPDH, Rubisco) were used, respectively; (c) expression level of NcNCED3 under cold treatment. Most stable reference genes (SAMDC, RPS25) and least stable reference genes (TUA2, EF-1-α) were used, respectively; (d) expression level of NcNCED3 under drought treatment. Most stable reference genes (SAMDC, RPS25) and least stable reference genes (GAPDH, Rubisco) recommended by BestKeeper were used, respectively. The error bars represent the mean of three biological replicates ± SD.