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High-Quality RNA Extraction and Evaluation of Reference Genes for qPCR Assay of Pinus sylvestris L. Trunk Tissues

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Abstract

Scots pine (Pinus sylvestris L.) is a species of tree with heartwood (HW), which forms during aging of sapwood (SW). Due to a clear-cut border between SW and HW, P. sylvestris should be used as a model woody plant for studying patterns of HW formation. Currently, molecular genetic methods are often used to study the processes of trunk tissues’ formation in woody plants. A feature of coniferous trees’ trunk tissues is a high content of secondary metabolites, a low content of nucleic acids, and potential partial degradation of RNA. In this work, the authors discuss the choice of the most successful method for extracting high-quality RNA for real-time PCR (RT-PCR) in P. sylvestris trunk tissues along the radial vector “conductive phloem/cambial zone–differentiating xylem–exterior part of SW (one to two annual rings)–interior part of SW (one to two annual rings afore transition zone (TZ))–TZ (two annual rings afore HW)” for reproducible RT-PCR data. The expression stability of six potential reference genes (Actin1, α-Tubulin, β-Tubulin, Ef1a, GAPDH, UBQ) was assessed in all describe tissues. Differences in expression levels of target genes are shown by data normalization using reference genes with different stability of expression.

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ACKNOWLEDGMENTS

The studies were carried out on the scientific equipment of the Center for Collective Use of the Federal Research Center Karelian Scientific Center, Russian Academy of Sciences.

Funding

The work was carried out within the framework of the state task of the Forest Institute (a separate subdivision of the Federal State Budgetary Institution of Science of the Federal Research Center Karelian Scientific Center, Russian Academy of Sciences) Comprehensive Study of the Productivity Factors of Taiga Forests (2021–2025, leader A.M. Kryshen, Ministry of Education and Science of Russia, 0185-2021-0018), state registration number 121061500082-2, with partial financial support from the Russian Science Foundation (grant 21-14-00204 (2021–2023, leader N.A. Galibina), phylogenetic analysis of reference and target genes).

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Authors and Affiliations

  1. Forest Research Institute of the Karelian Research Center, Russian Academy of Sciences, Petrozavodsk, Russia

    Yu. L. Moshchenskaya, N. A. Galibina, M. A. Korzhenevskiy, O. V. Chirva, T. V. Tarelkina & K. M. Nikerova

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  1. Yu. L. Moshchenskaya
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  2. N. A. Galibina
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  3. M. A. Korzhenevskiy
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  4. O. V. Chirva
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  5. T. V. Tarelkina
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  6. K. M. Nikerova
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Correspondence to Yu. L. Moshchenskaya.

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Moshchenskaya, Y.L., Galibina, N.A., Korzhenevskiy, M.A. et al. High-Quality RNA Extraction and Evaluation of Reference Genes for qPCR Assay of Pinus sylvestris L. Trunk Tissues. Russ J Dev Biol 54, 24–36 (2023). https://doi.org/10.1134/S1062360423010095

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