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

Yu. L. Moshchenskaya; Мощенская Ю. Л.; N. A. Galibina; Галибина Н. А.; M. A. Korzhenevskiy; Корженевский М. А.; T. V. Tarelkina; Тарелкина Т. В.; K. M. Nikerova; Никерова К. М.; O. V. Chirva; Чирва О. В.

doi:10.31857/S0475145023010093

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

Authors: Moshchenskaya Y.L.¹, Galibina N.A.¹, Korzhenevskiy M.A.¹, Tarelkina T.V.¹, Nikerova K.M.¹, Chirva O.V.¹
Affiliations:
1. Forest Research Institute of the Karelian Research Centre of the Russian Academy of Sciences
Issue: Vol 54, No 1 (2023)
Pages: 27-40
Section: Original study articles
URL: https://journal-vniispk.ru/0475-1450/article/view/137042
DOI: https://doi.org/10.31857/S0475145023010093
EDN: https://elibrary.ru/FRMYSQ
ID: 137042

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Abstract
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Abstract

Scots pine (Pinus sylvestris L.) is a species of tree with heartwood (HW), which is forming during aging of sapwood (SW). Due to 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 used to study the processes of trunk tissues formation in woody plants often. A feature of trunk tissues of coniferous trees is a high content of secondary metabolites, a low content of nucleic acids, and potential partial degradation of RNA. In this work we discuss the choice of most successful method for extraction a 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 (1–2 annual rings) – interior part of SW (1–2 annual rings afore transition zone (TZ)) – TZ (2 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.

Keywords

Pinus sylvestris, RNA extraction, normalization, RT-PCR, reference genes, gene expression

References

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