| Summary: | <p>Abstract</p> <p>Background</p> <p>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 <it>Populus</it>, 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 <it>Populus</it> 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 <it>Populus</it> genotypes (<it>P. trichocarpa</it> “Nisqually 1” and <it>P. tremula</it> x <it>P. alba</it> 717 1-B4) following MIQE guidelines.</p> <p>Results</p> <p>We evaluated gene expression stability in shoot tips, young leaves, mature leaves and bark tissues from <it>P. trichocarpa</it> and <it>P. tremula. x P. alba</it> grown under long-day (LD), short-day (SD) or SD plus low-temperatures conditions. Gene expression data were analyzed for stable reference genes among <it>18S rRNA</it>, <it>ACT2</it>, <it>CDC2</it>, <it>CYC063</it>, <it>TIP4-like</it>, <it>UBQ7</it>, <it>PT1</it> and <it>ANT</it> using two software packages, geNorm<sup>PLUS</sup> and BestKeeper. GeNorm<sup>PLUS</sup> ranked <it>TIP4-like</it> and <it>PT1</it> among the most stable genes in most genotype/tissue combinations while BestKeeper ranked <it>CDC2</it> and <it>ACT2</it> among the most stable genes.</p> <p>Conclusions</p> <p>This is the first comprehensive evaluation of reference genes in two important <it>Populus</it> genotypes and the only study in <it>Populus</it> 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 <it>Populus</it> species and genotypes. These results provide a valuable resource for the <it>Populus</it> research community.</p>
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