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Selection of suitable reference genes for quantitive real-time PCR normalization in Miscanthus lutarioriparia

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Abstract

Miscanthus lutarioriparia, which is found widespread in China, has attracted great attention as a most potential bioenergy plant for years. The quantitative real time PCR (RT-qPCR) has appeared as a sensitive and powerful technique to measure gene expression in living organisms during different development stages. In this study, we evaluated ten candidate genes, including 25S ribosomal RNA gene (25S rRNA), actin1 gene (ACT1), carotenoid-binding protein 20 gene (CBP20), glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH), Ubiquitin gene (UBQ), eukaryotic elongation factor 1-αgene (eEF-1α), α-tubulin gene (α-TUB), β-tubulin gene (β-TUB), eukaryotic translation initiation factor 4α-1 gene (eIF-4α) and NAC domain protein gene(NAC) in a series of 30 M. lutarioriparia samples followed by statistical algorithms geNorm and Normfinder to analyze the gene expression stability. The results indicated that eIF-4αand UBQ were the most stable expressed genes while CBP20 showed as the least stable among all the samples. Based on above research, we recommend that at least two top-ranked reference genes should be employed for expression data normalization. The best genes selected in this study will provide a starting point to select reference genes in the future in other tissues and under other experimental conditions in this energy crop candidate.

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Abbreviations

RT-qPCR:

Quantitative real-time reverse transcription polymerase chain reaction

25S rRNA :

25S ribosomal RNA

ACT1 :

Actin 1

CBP20 :

Carotenoid-binding protein 20

GAPDH :

Glyceraldehyde-3-phosphate dehydrogenase

UBQ :

Ubiquitin

eEF-1α :

Elongation factor-1α

αTUB :

α Tubulin

βTUB :

β Tubulin

eIF-4α :

Eukaryotic translation initiation factor 4α

NAC :

NAC domain protein

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Acknowledgements

This work was financially supported by the National Natural Science Foundation of China (Grant No. 31571740) and the National High-Tech R&D Program (Grant No. 2012AA101801), Natural Science Foundation of Hubei Province (Grant No. 2013CFA103).

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Authors and Affiliations

  1. State Key Laboratory of Hybrid Rice, Hubei Lotus Engineering Center College of Life Sciences, Wuhan University, Wuhan, 430072, People’s Republic of China

    Teng Cheng, Fenglin Zhu, Jiajing Sheng, Lingling Zhao, Fasong Zhou, Zhongli Hu & Ying Diao

  2. School of Life Sciences Nantong University, Nantong, 226019, People’s Republic of China

    Jiajing Sheng

  3. School of Chemistry, Chemical Engineering and Life Science, Wuhan University of Technology, Wuhan, 430070, People’s Republic of China

    Surong Jin

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  1. Teng Cheng
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Correspondence to Ying Diao or Surong Jin.

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11033_2019_4910_MOESM1_ESM.eps

Supplementary material 1 Expression profile of candidate reference genes in different tissues of M. lutarioriparius Ct values in different part of M. lutarioriparius were counted for expression analysis. Red point represent the mean value of the Ct at a certain tissue. Whisskers represent the range of standard errors (EPS 8529 kb)

Supplementary material 2 (DOC 37 kb)

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Cheng, T., Zhu, F., Sheng, J. et al. Selection of suitable reference genes for quantitive real-time PCR normalization in Miscanthus lutarioriparia. Mol Biol Rep 46, 4545–4553 (2019). https://doi.org/10.1007/s11033-019-04910-8

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