Chin Bull Bot ›› 2015, Vol. 50 ›› Issue (6): 754-764.doi: 10.11983/CBB14173

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Validation of Reference Genes for Quantitative RT-PCR Analysis in Soybean Root Tissue under Salt Stress

Qiong Jiang1,2, Youning Wang1, Lixiang Wang1,2, Zhengxi Sun1,2, Xia Li1*   

  1. 1Center of Agricultural Research Resources, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Shijiazhuang 050021, China
    2University of Chinese Academy of Sciences, Beijing 100049, China
  • Received:2014-09-22 Accepted:2015-01-14 Online:2015-09-06 Published:2015-11-01
  • Contact: Li Xia E-mail:xli@genetics.ac.cn
  • About author:

    ? These authors contributed equally to this paper

Abstract:

Quantitative RT-PCR (qRT-PCR) is a robust and commonly used technique to study gene expression. However, the reference genes and microRNA (miRNA) that can be used in soybean molecular research, in particular the stress responsive genes and miRNA in roots, are not well characterized. In this study, we systematically analyzed the expression stability of eight widely used reference genes (ACT, ACT2/7, CYP2, ELF1A, ELF1B, F-Box, TUA and UBC2) and miRNA internal references (U6, miR1515a, miR1520c, miR1520d, miR171a, and miR171b) in root tissues treated with different NaCl concentrations at specified times. The Δ-Ct method, Bestkeeper 1.0, NormFinder 0.953 and Genorm 3.5 were used to analyze the data, then the web-based tool RefFinder was used to integrate the results obtained. ELF1B/CYP2 and miR1515a/U6 were the most suitable genes for qRT-PCR analysis of soybean gene and miRNA expression analysis under salt stress, respectively. The highly ranked reference genes and miRNAs identified from this study can be used for detecting differences in expression rates of genes and miRNAs in response to salt stress in soybean.

Figure 1

PCR products of fourteen reference genes. M: DNA marker; 1: ACT; 2: ACT2/7; 3: CYP2; 4: ELF1A; 5: ELF1B; 6: F-Box; 7: TUA; 8: UBC2; 9: U6; 10: miR1515a; 11: miR1520c; 12: miR1520d; 13: miR171a; 14: miR171b"

Table 2

Stability ranking of candidate reference genes"

Method Stability (high→low)
1 2 3 4 5 6 7 8
Δ-CT methods CYP2 ACT ELF1B UBC2 ACT2/7 ELF1A F-Box TUA
Bestkeeper ELF1B TUA UBC2 CYP2 ACT ACT2/7 ELF1A F-Box
NormFinder CYP2 ACT ELF1B UBC2 ELF1A ACT2/7 F-Box TUA
Genorm ELF1B /UBC2 CYP2 ACT ACT2/7 F-Box ELF1A TUA
Recommended
comprehensive ranking
ELF1B CYP2 UBC2 ACT ACT2/7 TUA ELF1A F-Box

Figure 2

Real-time PCR melting curves of eight reference genes in soybean roots under salt stress(A) ACT; (B) ACT2/7; (C) CYP2; (D) ELF1A; (E) ELF1B; (F) F-Box; (G) TUA; (H) UBC2"

Figure 3

Real-time PCR melting curves of six miRNAs in soybean roots under salt stress(A) U6; (B) miR1515a; (C) miR1520c; (D) miR1520d; (E) miR171a; (F) miR171b"

Table 3

Stability ranking of candidate miRNA"

Method Stability (high→low)
1 2 3 4 5 6
Δ-CT methods miR1520c miR1515a miR171a U6 miR1520d miR171b
Bestkeeper miR1515a miR1520c U6 miR171a miR1520d miR171b
NormFinder miR1520c miR1515a miR171a U6 miR1520d miR171b
Genorm U6/miR1520c miR1515a miR171a miR1520d miR171b
Recommended
comprehensive ranking
miR1520c miR1515a U6 miR171a miR1520d miR171b

Figure 4

Expression level of all candidate reference genes in soybean roots under salt stress. 1-14 see Figure 1."

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