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. 2018 Dec 31;24(1):136.
doi: 10.3390/molecules24010136.

Selection of Reference Genes for qRT-PCR Analysis in Lentinula edodes after Hot-Air Drying

Shuangshuang Gao  1 , Gangzheng Wang  2 , Zhicheng Huang  3 , Xiaoyu Lei  4 , Yinbing Bian  5 , Ying Liu  6 , Wen Huang  7
Affiliations

Selection of Reference Genes for qRT-PCR Analysis in Lentinula edodes after Hot-Air Drying

Shuangshuang Gao et al. Molecules. .

Abstract

Volatile sulfur compounds gradually develop in Lentinula edodes after hot-air drying, and many genes are involved in the generation of these sulfur compounds. The expression stability of reference genes may vary in a particular experimental treatment when analyzing their expressions by quantitative real-time polymerase chain reaction (qRT-PCR). In this study, the expression profile of 17 candidate genes was assessed in L. edodes under treatment at 50 °C for 0, 1, 2, and 3 h, and the expression stability of each reference gene was analyzed by three statistical algorithms, including geNorm, NormFinder, and BestKeeper. Results indicated that the two optimal reference genes for mycelium and fruiting body were CAC and DAHP as well as CAC and NUP, respectively. Additionally, CAC and DAHP were found to be the two most stable reference genes across the mycelium and fruiting body set. Our results will provide a genetic foundation for further research on the metabolism genes of sulfur compounds in L. edodes.

Keywords: Lentinula edodes; hot-air drying; qRT-PCR; reference genes; volatile sulfur compounds.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Figure 1
Figure 1
Variation in qRT-PCR values of candidate reference genes.
Figure 2
Figure 2
Expression stability and ranking of candidate reference genes determined by geNorm. (A) mycelium data set; (B) fruiting body data set; (C) all-sample data set; (D) pairwise variation (Vn/Vn + 1) for the optimal number of reference genes.
Figure 3
Figure 3
Validation of the selected reference genes. Relative expression of DnaJ and LOX in mycelium (A, B) and fruiting body (C, D). In mycelium, CAC and DAHP were the most stable candidate reference genes, and MSH3 was the least stable candidate reference gene. In fruiting body, CAC and NUP were the most stable candidate reference genes, and AP2A was the least stable candidate reference gene. ACT, commonly used as endogenous control, was also adopted. Data are shown as mean ± standard deviation (n = 3).
Figure 4
Figure 4
Relative expression of different genes during hot-air drying. Mycelia was treated with 0, 0.5, 1, 1.5, 2, 3, 4, and 5 h (A). Fruiting body was treated with 0, 1, 2, 3, 4, 5, 6, and 7 h (B).
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