Abstract
Reverse transcription-quantitative real-time PCR (RT-qPCR) is a widely used technique for gene expression analysis. The reliability of this method depends largely on the suitable selection of stable reference genes for accurate data normalization. Hypericum perforatum L. (St. John's wort) is a field growing plant that is frequently exposed to a variety of adverse environmental stresses that can negatively affect its productivity. This widely known medicinal plant with broad pharmacological properties (anti-depressant, anti-tumor, anti-inflammatory, antiviral, antioxidant, anti-cancer, and antibacterial) has been overlooked with respect to the identification of reference genes suitable for RT-qPCR data normalization. In this study, 11 candidate reference genes were analyzed in H. perforatum plants subjected to cold and heat stresses. The expression stability of these genes was assessed using GeNorm, NormFinder and BestKeeper algorithms. The results revealed that the ranking of stability among the three algorithms showed only minor differences within each treatment. The best-ranked reference genes differed between cold- and heat-treated samples; nevertheless, TUB was the most stable gene in both experimental conditions. GSA and GAPDH were found to be reliable reference genes in cold-treated samples, while GAPDH showed low expression stability in heat-treated samples. 26SrRNA and H2A had the highest stabilities in the heat assay, whereas H2A was less stable in the cold assay. Finally, AOX1, AOX2, CAT1 and CHS genes, associated with plant stress responses and oxidative stress, were used as target genes to validate the reliability of identified reference genes. These target genes showed differential expression profiles over time in treated samples. This study not only is the first systematic analysis for the selection of suitable reference genes for RT-qPCR studies in H. perforatum subjected to temperature stress conditions, but may also provide valuable information about the roles of genes associated with temperature stress responses.
Highlights
Gene expression analysis has been widely used as a method to study the complex signaling and metabolic pathways underlying cellular and developmental processes in biological organisms, including plants
Despite the pharmacological interest of the compounds produced by H. perforatum, very limited genomic information is available in NCBI
For this study, a significant number of the genes selected had been recently isolated by members of our research group (GAPDH: EU301783, GSA: KJ624985, H2A: EU034009, short-chain dehydrogenase/reductase (SDR): EU034010, TUB: KJ669725, AOX1: EU330415, AOX2: EU330413)
Summary
Gene expression analysis has been widely used as a method to study the complex signaling and metabolic pathways underlying cellular and developmental processes in biological organisms, including plants. The analysis of gene expression has been performed by using different methods such as, northern blotting, ribonuclease protection assay, reverse transcriptionpolymerase chain reaction (RT-PCR), reverse transcription-quantitative real-time PCR (RT-qPCR), DNA microarrays [5], and generation sequencing (NGS) technologies [6]. These last three technologies in particular have gained a wider appeal for the quantification of gene expression. Several variables need to be controlled to obtain reliable quantitative expression measures by RT-qPCR These include variations in initial sample quantity, RNA recovery, RNA integrity, efficiency of cDNA synthesis, and differences in the overall transcriptional activity of the tissues or cells analyzed [11]. The identification of stable RGs in various experimental designs will contribute to have more accurate and reliable gene expression data
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