Abstract
To accurately quantify gene expression using quantitative PCR amplification, it is vital that one or more ideal internal control genes are used to normalize the samples to be compared. Ideally, the expression level of those internal control genes should vary as little as possible between tissues, developmental stages and environmental conditions. In this study, 32 candidate genes for internal control were obtained from the analysis of nine independent experiments which included 333 Affymetrix GeneChip Wheat Genome arrays. Expression levels of the selected genes were then evaluated by quantitative real-time PCR with cDNA samples from different tissues, stages of development and environmental conditions. Finally, fifteen novel internal control genes were selected and their respective expression profiles were compared using NormFinder, geNorm, Pearson correlation coefficients and the twofold-change method. The novel internal control genes from this study were compared with thirteen traditional ones for their expression stability. It was observed that seven of the novel internal control genes were better than the traditional ones in expression stability under all the tested cDNA samples. Among the traditional internal control genes, the elongation factor 1-alpha exhibited strong expression stability, whereas the 18S rRNA, Alpha-tubulin, Actin and GAPDH genes had very poor expression stability in the range of wheat samples tested. Therefore, the use of the novel internal control genes for normalization should improve the accuracy and validity of gene expression analysis.
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Acknowledgment
The authors thank two anonymous reviewers for their constructive suggestions. This work was supported by the National Basic Research Program of China (973 Program 2010CB1344400 and 2009CB118304) and China Transgenic Research Program (2011ZX08002).
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Xiang-Yu Long and Ji-Rui Wang contributed equally to this paper.
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11103_2010_9666_MOESM3_ESM.tif
Supp Fig. 1 Expression stability and ranking of candidate internal control genes were calculated by geNorm and NormFinder in group one (a), group two (b) and group three (c). The red-colored bars present the average expression stability value (M) calculated by geNorm, and the blue-colored ones present the stability value calculated by NormFinder. Lower stability value of expression indicates more stable expression (TIFF 764 kb)
11103_2010_9666_MOESM4_ESM.tif
Supp Fig. 2 Correlation of ranking from NormFinder and geNorm was evaluated for all candidate internal control genes in group one (TIFF 2406 kb)
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Supp Fig. 3 The optimal number of control genes was determined for normalization in group one. (a) The geNorm analyzes the pairwise variation between internal control genes to determine the optimal number. (b).The NormFinder calculates the accumulated standard deviation of candidate internal control genes to determine the optimal number (TIFF 767 kb)
11103_2010_9666_MOESM6_ESM.tif
Supp Fig. 4 The optimal number of control genes was determined by twofold-method in group one. The ratio was calculated by comparing the expression of genes with each other. The black-colored bars represent the log2 mean of ratio of gene expression. The white-colored bars represent the mean of the log2 ratio of gene expression (TIFF 1953 kb)
11103_2010_9666_MOESM7_ESM.tif
Supp Fig. 5 Expression levels of candidate internal control genes were tested using the qRT–PCR quantification cycle values (Cq). In general, transcripts fell into several categories based on their expression strength, which could be divided into groups: group A with high RNA transcription levels (mean Cq < 20), group B with medium RNA transcription levels (20 < mean Cq < 25) and group C with low RNA transcription levels (mean Cq > 25). The group A genes included two candidate internal control genes, and the group B and C genes included fifteen candidate internal control genes respectively. The red-dot represents the internal control genes expression level (Cq) in each samples and the black square represents the mean expression level of internal control genes (arithmetical mean of Cqs) (TIFF 704 kb)
11103_2010_9666_MOESM8_ESM.tif
Supp Fig. 6 Expression stability and ranking of novel and traditional internal control genes were calculated by geNorm and NormFinder in group one. The red-colored bars present the average expression stability value (M) calculated by geNorm, and the blue-colored presents the stability value calculated by NormFinder. Lower stability value of expression indicates more stable expression (TIFF 1473 kb)
11103_2010_9666_MOESM9_ESM.tif
Supp Fig. 7 The optimal number of control genes for normalization was determined in group one. (a) The geNorm analyzes the pairwise variation between novel and traditional internal control genes to determine the optimal number. (b).The NormFinder calculates the accumulated standard deviation of novel and traditional internal control genes to determine the optimal number (TIFF 2020 kb)
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Long, XY., Wang, JR., Ouellet, T. et al. Genome-wide identification and evaluation of novel internal control genes for Q-PCR based transcript normalization in wheat. Plant Mol Biol 74, 307–311 (2010). https://doi.org/10.1007/s11103-010-9666-8
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DOI: https://doi.org/10.1007/s11103-010-9666-8