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The use of miRNAs as reference genes for miRNA expression normalization during Lilium somatic embryogenesis by real-time reverse transcription PCR analysis

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Abstract

Expression profiling of miRNAs has the ability to reveal the essence of somatic embryogenesis (SE). qRT-PCR is one of the most commonly used techniques for dynamic miRNA detection but requires optimal reference genes for data reliability. This is the first report on reference gene validation for miRNA expression normalization in Lilium (Lilium pumilum DC. Fisch. and Lilium davidii var. unicolor). In this study, seventeen miRNAs together with two snRNAs (U4, U6), one rRNA (5S rRNA) and three protein-coding genes (FP, ACT, GAPDH) were selected as reference candidates, and their expression stability was validated by qRT-PCR among eleven develo** SE cultures in two lilies. Four normalization algorithms, including geNorm, BestKeeper, NormFinder and RefFinder, were also used to evaluate the stability of the reference candidates. For Lilium pumilum DC. Fisch., lpu-miR159a was the optimal reference gene during SE, followed by lpu-miR408b, while U6 was the least stable reference candidate. For Lilium davidii var. unicolor, FP presented greater stability than did half of the miRNA candidates, but the best reference gene was lda-miR162, followed by lda-miR159a. Further analysis of the expression level of miR156 and miR529 was used to evaluate the validity of the reference genes in both lilies. In general, miRNAs are superior to common protein-coding genes and snRNAs / rRNAs as reference genes for miRNA expression normalization during Lilium SE, and the most suitable reference miRNA is different between two species in the same Lilium genus. This is a pioneer study using suitable miRNAs as reference genes in Lilium and constitutes a small but essential step for the further exploration of miRNA function in Lilium, thus offering valuable references for other plants.

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Acknowledgements

This project was supported by the National Natural Science Foundation of China [grant number 31471897; 31672179] and the Program for Excellent Talents in the University of Liaoning province, China [grant number LR2013029].

Author contributions

JZ and HMS conceived and designed the experiment. MZG and BYX contributed reagents and materials. CXW designed the primers for qRT-PCR. JZ, MZG, BYX and NNJ performed RNA extraction and qRT-PCR. JZ and JXW analyzed the data. JZ and HMS wrote and revised the manuscript. All of the authors read and approved the final manuscript.

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  1. Key Laboratory of Protected Horticulture of Education Ministry and Liaoning Province, College of Horticulture, Shenyang Agricultural University, Shenyang, People’s Republic of China

    **g Zhang, MeiZhu Gai, BingYang Xue, NaNa Jia, Chun**a Wang, ****a Wang & HongMei Sun

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Zhang, J., Gai, M., Xue, B. et al. The use of miRNAs as reference genes for miRNA expression normalization during Lilium somatic embryogenesis by real-time reverse transcription PCR analysis. Plant Cell Tiss Organ Cult 129, 105–118 (2017). https://doi.org/10.1007/s11240-016-1160-9

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