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

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. [ABSTRACT FROM AUTHOR]