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. 2019 Jun 13;10(6):451.
doi: 10.3390/genes10060451.

Selection of Suitable Reference Genes for RT-qPCR Gene Expression Analysis in Siberian Wild Rye (Elymus sibiricus) under Different Experimental Conditions

Junchao Zhang  1 , Wengang Xie  2 , Xinxuan Yu  3 , Zongyu Zhang  4 , Yongqiang Zhao  5 , Na Wang  6 , Yanrong Wang  7
Affiliations

Selection of Suitable Reference Genes for RT-qPCR Gene Expression Analysis in Siberian Wild Rye (Elymus sibiricus) under Different Experimental Conditions

Junchao Zhang et al. Genes (Basel). .

Abstract

Elymus sibiricus, which is a perennial and self-pollinated grass, is the typical species of the genus Elymus, which plays an important role in forage production and ecological restoration. No reports have, so far, systematically described the selection of optimal reference genes for reverse transcriptase quantitative real-time polymerase chain reaction (RT-qPCR) analysis in E. sibiricus. The goals of this study were to evaluate the expression stability of 13 candidate reference genes in different experimental conditions, and to determine the appropriate reference genes for gene expression analysis in E. sibiricus. Five methods including Delta Ct (ΔCt), BestKeeper, NormFinder, geNorm, and RefFinder were used to assess the expression stability of 13 potential reference genes. The results of the RefFinder analysis showed that TBP2 and HIS3 were the most stable reference genes in different genotypes. TUA2 and PP2A had the most stable expression in different developmental stages. TBP2 and PP2A were suitable reference genes in different tissues. Under salt stress, ACT2 and TBP2 were identified as the most stable reference genes. ACT2 and TUA2 showed the most stability under heat stress. For cold stress, PP2A and ACT2 presented the highest degree of expression stability. DNAJ and U2AF were considered as the most stable reference genes under osmotic stress. The optimal reference genes were selected to investigate the expression pattern of target gene CSLE6 in different conditions. This study provides suitable reference genes for further gene expression analysis using RT-qPCR in E. sibiricus.

Keywords: Elymus sibiricus; experimental conditions; expression stability; reference genes; reverse transcriptase quantitative real-time polymerase chain reaction (RT-qPCR).

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Primer specificity and amplicon size of 13 candidate reference genes. (A) Melting curves of 13 candidate reference genes exhibiting single peaks. (B) Agarose gel electrophoresis (2%) showing specific amplification products of expected size using Real Time-qPCR. M: 500 bp marker. 1–13: ACT2, CYP19, DNAJ, eIF-3A, eIF-3C, GAPDH, HIS3, PP2A, TBP2, TEF2, TUA2, TUB3, and U2AF.
Figure 2
Figure 2
RT-qPCR Cq values for all candidate reference genes in all E. sibiricus samples. Whisker caps, boxes, lines, and square boxes represent maximum/minimum, 25/75 percentiles, median, and mean, respectively.
Figure 3
Figure 3
Average expression stability values (M) of 13 candidate reference genes under different conditions calculated by geNorm. (A) Different genotypes, (B) different developmental stages, (C) different tissues, (D) salt stress, (E) heat stress, (F) cold stress, (G) osmotic stress, and (H) all samples.
Figure 4
Figure 4
Pairwise variation (V) of 13 candidate reference genes under various experimental conditions calculated by geNorm.
Figure 5
Figure 5
Expression stability of E. sibiricus potential reference genes calculated using NormFinder. (A) Different genotypes, (B) different developmental stages, (C) different tissues, (D) salt stress, (E) heat stress, (F) cold stress, (G) osmotic stress, and (H) all samples. Low to high expression stability is represented over a spectrum from red to blue, respectively.
Figure 6
Figure 6
Expression stability of candidate reference genes calculated by BestKeeper. (A) Different genotypes, (B) different developmental stages, (C) different tissues, (D) salt stress, (E) heat stress, (F) cold stress, (G) osmotic stress, and (H) all samples. Low to high expression stability is represented over a spectrum from red to blue, respectively.
Figure 7
Figure 7
Expression stability for 13 candidate reference genes calculated via Delta Ct. (A) Different genotypes, (B) different developmental stages, (C) different tissues, (D) salt stress, (E) heat stress, (F) cold stress, (G) osmotic stress, and (H) all samples. Low to high expression stability is represented over a spectrum from red to blue, respectively.
Figure 8
Figure 8
The relative expression level of target gene CSLE6 in various experimental conditions. The most two stable reference genes and the most unstable reference genes in different conditions were selected for expression normalization. (A) Different genotypes, (B) different developmental stages, (C) different tissues, (D) salt stress, (E) heat stress, (F) cold stress, and (G) osmotic stress. Different letters in the same sample represent a significant difference among three reference genes at the 0.05 level. Error bars indicate standard deviation.
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