Abstract
The vertical segments of Populus stems are an ideal experimental system for analyzing the gene expression patterns involved in primary and secondary growth during wood formation. Suitable internal control genes are indispensable to quantitative real time PCR (qRT-PCR) assays of gene expression. In this study, the expression stability of eight candidate reference genes was evaluated in a series of vertical stem segments of Populus tomentosa. Analysis through software packages geNorm, NormFinder and BestKeeper showed that genes ribosomal protein (RP) and tubulin beta (TUBB) were the most unstable across the developmental stages of P. tomentosa stems, and the combination of the three reference genes, eukaryotic translation initiation factor 5A (eIF5A), Actin (ACT6) and elongation factor 1-beta (EF1-beta) can provide accurate and reliable normalization of qRT–PCR analysis for target gene expression in stem segments undergoing primary and secondary growth in P. tomentosa. These results provide crucial information for transcriptional analysis in the P. tomentosa stem, which may help to improve the quality of gene expression data in these vertical stem segments, which constitute an excellent plant system for the study of wood formation.
Highlights
Gene expression analysis always provides important clues regarding gene function in plant growth and development and response to environmental changes
Reference genes with stable expression levels are indispensable to normalizing the expression levels of the target genes in quantitative real time PCR (qRT-PCR) analysis in order to control the variation caused by the differences in initial RNA amount, RNA integrity and purity, and enzyme efficiency [1, 2]
Validation of reference genes is highly specific to particular experimental models, and it is a crucial step in the initiation of analysis of RNA transcription levels by qRT-PCR in a series of specific tissues with a given developmental period [2, 6, 7, 9, 10]
Summary
Gene expression analysis always provides important clues regarding gene function in plant growth and development and response to environmental changes. There are several housekeeping genes, which are involved in basic cellular and metabolic processes that are usually used as reference genes in qRT-PCR assays These include ribosomal protein (RP), tubulin (TUB), actin (ACT), 18S rRNA (18S), elongation factor (EF), eukaryotic translation initiation factor (eIF), ubiquitin (UBQ), and histone H3 (HIS3) [3,4,5,6]. Validation of reference genes is highly specific to particular experimental models, and it is a crucial step in the initiation of analysis of RNA transcription levels by qRT-PCR in a series of specific tissues with a given developmental period [2, 6, 7, 9, 10]
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