Abstract
Despite the unshakable status of reverse transcription-quantitative PCR in gene expression analysis, it has certain disadvantages, including that the results are highly dependent on the reference genes selected for data normalization. Since inappropriate endogenous control genes will lead to inaccurate target gene expression profiles, the validation of suitable internal reference genes is essential. Given the increasing interest in functional genes and genomics of Populus euphratica, a desert poplar showing extraordinary adaptation to salt stress, we evaluated the expression stability of ten candidate reference genes in P. euphratica roots, stems, and leaves under salt stress conditions. We used five algorithms, namely, ΔCt, NormFinder, geNorm, GrayNorm, and a rank aggregation method (RankAggreg) to identify suitable normalizers. To support the suitability of the identified reference genes and to compare the relative merits of these different algorithms, we analyzed and compared the relative expression levels of nine P. euphratica functional genes in different tissues. Our results indicate that a combination of multiple reference genes recommended by GrayNorm algorithm (e.g., a combination of Actin, EF1α, GAPDH, RP, UBQ in root) should be used instead of a single reference gene. These results are valuable for research of gene identification in different P. euphratica tissues.
Highlights
Gene expression characterization at the steady-state mRNA level is an important step in many plant biological processes
The analyses show that the different reference genes identified by different methods had a variable impact on gene of interest data, and that a reliable set of reference genes should be selected based on the highest possible accuracy of reverse transcription-quantitative polymerase chain reaction (RT-qPCR) results
[26] Genes used to support the suitability of the identified reference genes; d GenBank accession number; e The RT-qPCR efficiency was determined by standard curve method and confirmed by LinReg PCR program
Summary
Gene expression characterization at the steady-state mRNA level is an important step in many plant biological processes. Previous studies of P. euphratica have mainly focused on analyses of salt-responsive genes [26,30,34,35], miRNAs [36,37], or salt-related transcriptome sequencing [26,38,39] In these studies, the genes up-regulated or down-regulated by salt stress are important and should be identified using validated reference genes by RT-qPCR. The analyses show that the different reference genes identified by different methods had a variable impact on gene of interest data, and that a reliable set of reference genes should be selected based on the highest possible accuracy of RT-qPCR results This approach can be applied to future studies of gene expression profiles in P. euphratica
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