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Selection and validation of suitable reference genes for miRNA expression normalization by quantitative RT-PCR in citrus somatic embryogenic and adult tissues

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Abstract

miRNAs have recently been reported to modulate somatic embryogenesis (SE), a key pathway of plant regeneration in vitro. For expression level detection and subsequent function dissection of miRNAs in certain biological processes, qRT-PCR is one of the most effective and sensitive techniques, for which suitable reference gene selection is a prerequisite. In this study, three miRNAs and eight non-coding RNAs (ncRNA) were selected as reference candidates, and their expression stability was inspected in developing citrus SE tissues cultured at 20, 25, and 30 °C. Stability of the eight non-miRNA ncRNAs was further validated in five adult tissues without temperature treatment. The best single reference gene for SE tissues was snoR14 or snoRD25, while for the adult tissues the best one was U4; although they were not as stable as the optimal multiple references snoR14 + U6 for SE tissues and snoR14 + U5 for adult tissues. For expression normalization of less abundant miRNAs in SE tissues, miR3954 was assessed as a viable reference. Single reference gene snoR14 outperformed multiple references for the overall SE and adult tissues. As one of the pioneer systematic studies on reference gene identification for plant miRNA normalization, this study benefits future exploration on miRNA function in citrus and provides valuable information for similar studies in other higher plants.

Key message Three miRNAs and eight non-coding RNAs were tested as reference candidates on developing citrus SE tissues. Best single references snoR14 or snoRD25 and optimal multiple references snoR14 + U6, snoR14 + U5 were identified.

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Abbreviations

miRNA:

microRNA

ncRNA:

Non-coding RNA

qRT-PCR:

Quantitative reverse transcription-polymerase chain reaction

SE:

Somatic embryogenesis

NEC:

Non-embryogenic callus

EC:

Embryogenic callus

E1/2/3/4:

Embryogenic callus induced for somatic embryo for 1/2/3/4 weeks

GE:

Globular embryo

HE:

Heart embryo

CE:

Cotyledonary embryo

Ct:

Cycle threshold

Csi:

Citrus sinensis

TPM:

Times per million

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Acknowledgments

This research was financially supported by the Ministry of Science & Technology of China (nos. 2011CB100606, 2011AA100205), the National NSF of China and the Doctoral Research Fund of Ministry of Education of China (no. 20100146110008). The authors thank Dr. Bo Zheng (Huazhong Agric. Univ.) for reading the manuscript.

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Authors and Affiliations

  1. Key Laboratory of Horticultural Plant Biology (Ministry of Education), National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan, 430070, China

    Shu-Jun Kou, Xiao-Meng Wu, Zheng Liu, Yuan-Long Liu, Qiang Xu & Wen-Wu Guo

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  1. Shu-Jun Kou
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  2. Xiao-Meng Wu
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  3. Zheng Liu
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  4. Yuan-Long Liu
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Correspondence to Wen-Wu Guo.

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Communicated by R. Rose.

S.-J. Kou and X.-M Wu contributed equally to this work.

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Kou, SJ., Wu, XM., Liu, Z. et al. Selection and validation of suitable reference genes for miRNA expression normalization by quantitative RT-PCR in citrus somatic embryogenic and adult tissues. Plant Cell Rep 31, 2151–2163 (2012). https://doi.org/10.1007/s00299-012-1325-x

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