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| The Selection of Endogenous Reference Genes in Phyllostachys edulis for qRT-PCR Analysis |
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Abstract Quantitative Real-time PCR (qRT-PCR) is a commonly-used method for studying gene expression currently, in which choosing appropriate reference genes for specific materials or particular conditions is a prerequisite for accurately analyzing relative expressions of target genes. This study took the roots, stems, leaves and bamboo shoots of Phyllostachys edulis as experimental materials and analyzed the expressions of 5 kinds of widely used reference genes - glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin (ACT), heat shock protein (HSP), 18S ribosomal RNA (18S rRNA) and eukaryotic initiation factor 4α (eIF-4α) in different tissues or organs by the aid of the qRT-PCR. Three programs including GeNorm, NormFinder and BestKeeper were used to determine the expression stability of these reference genes. The results showed that the most stable reference genes of P. edulis had many differences in the various experimental conditions. After the comparative analysis using GeNorm, NormFinder and BestKeeper, eIF-4α had the highest expression stability and was viewed as the corrective reference gene when the genes expression in different leaves of P. edulis were analyzed and compared by qRT-PCR. When the differences of genes expression were compared in different parts of bamboo shoots, ACT or 18S rRNA could be chosen as the corrective reference gene in different parts of bamboo shoots. In order to compare the differences of gene expression among the roots, stems and leaves of P. edulis, the expression stability of 18S rRNA was the best on GeNorm and BestKeeper and was second on NormFinder. Therefore, 18S rRNA could be chosen as corrective reference gene in different tissues of P. edulis. These findings were inconsistent with those of previous studies, which ACT was used as the only one reference gene. This study suggested that suitable reference genes should be selected on the basis of specific requirements, experiment conditions, and the characteristics of experimental material in practical applications. The results of this study would provide a theoretical basis for selecting the appropriate reference gene of P. edulis in the analysis of qRT-PCR.
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Received: 20 July 2017
Published: 14 February 2018
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