Evaluation of qPCR reference genes in two genotypes of Populus for use in photoperiod and low-temperature studies

ABSTRACT: BACKGROUND: Quantitative PCR (qPCR) is a widely used technique for gene expression analysis. A common normalization method for accurate qPCR data analysis involves stable reference genes to determine relative gene expression. Despite extensive research in the forest tree species Populus, there is not a resource for reference genes that meet the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) standards for qPCR techniques and analysis. Since Populus is a woody perennial species, studies of seasonal changes in gene expression are important towards advancing knowledge of this important developmental and physiological trait. The objective of this study was to evaluate reference gene expression stability in various tissues and growth conditions in two important Populus genotypes (P. trichocarpa "Nisqually 1" and P. tremula X P. alba 717 1-B4) following MIQE guidelines. RESULTS: We evaluated gene expression stability in shoot tips, young leaves, mature leaves and bark tissues from P. trichocarpa and P. tremula. x P. alba grown under long-day (LD), short-day (SD) or SD plus low-temperatures conditions. Gene expression data were analyzed for stable reference genes among 18S rRNA, ACT2, CDC2, CYC063, TIP4-like, UBQ7, PT1 and ANT using two software packages, geNormPLUS and BestKeeper. GeNormPLUS ranked TIP4-like and PT1 among the most stable genes in most genotype/tissue combinations while BestKeeper ranked CDC2 and ACT2 among the most stable genes. CONCLUSIONS: This is the first comprehensive evaluation of reference genes in two important Populus genotypes and the only study in Populus that meets MIQE standards. Both analysis programs identified stable reference genes in both genotypes and all tissues grown under different photoperiods. This set of reference genes was found to be suitable for either genotype considered here and may potentially be suitable for other Populus species and genotypes. These results provide a valuable resource for the Populus research community.     

Reference Gene Selection for qRT-PCR Normalization in <i>Iris germanica</i> L.

Quantitative real-time PCR (qPCR) is an effective and widely used method to analyze expression patterns of target genes. Selection of stable reference genes is a prerequisite for accurate normalization of target gene expression by qRT-PCR. In Iris germanica L., no studies have yet been published regarding the evaluation of potential reference genes. In this study, nine candidate reference genes were assessed at different flower developmental stages and in different tissues by four different algorithms (GeNorm, NormFinder, BestKeeper, and RefFinder). The results revealed that ACT11 (Actin 11) and EF1α (Elongation factor 1 alpha) were the most stable reference genes in different tissues, whereas TUA (Tubulin alpha) and UBC9 (Ubiquitin-protein ligase 9) were the most stable ones in different flower developmental stages. UBC9 and ACT11 were the most stable reference genes in all of the tested samples, while the SAMDC (S-Adenosylmethionine decarboxylase) showed the least stability. Finally, to validate the suitability of the selected reference genes, the relative expression level of IgTPS (beta-caryophyllene synthase) was assessed and highlighted the importance of suitable reference gene selection. This work constitutes the first systematic evaluation of potential reference genes in I. germanica and provides guidelines for future research on gene function and molecular mechanisms on I. germanica and related species.