Abstract
Accurate normalization of gene expression data is an absolute prerequisite to obtain reliable results in qPCR analysis. Oenanthe javanica, an aquatic perennial herb, belongs to the Oenanthe genus in Apiaceae family, with known medicinal properties. In the current study, O. javanica was subjected to hormone stimuli (gibberellin, salicylic acid, methyl jasmonate, and abscisic acid) and abiotic stresses (heat, cold, salt, and drought), and the expression of nine candidate reference genes (eIF-4α, ACT7, TIP41, GAPDH, SAND, EF-1α, PP2A, TBP, and TUB) was evaluated. Stability of the genes was assessed using geNorm, NormFinder and BestKeeper. All the genes presented distinct expression profiles under the experimental conditions analyzed. Under abiotic stress conditions, ACT7 and PP2A genes displayed the maximum stability; PP2A and SAND were the most stable genes under hormone stimuli. Even though PP2A gene was most stable across all the samples, individual analysis revealed changes in expression profile. To further validate the suitability of the reference genes identified in this study, the expression level of M6PR gene under salt treatment was studied. Based on our data, we propose that it is essential to normalize the target gene expression with specific reference genes under different experimental conditions for most accurate results. To our knowledge, this is the first systematic analysis for reference genes under abiotic stress and hormone stimuli conditions in O. javanica. This will be beneficial for future studies on O. javanica and other plants in Apiaceae family at molecular level.
Highlights
Due to its specificity, accuracy, efficiency and reproducibility, Quantitative real-time PCR has become the most prevalent method for quantifying transcript expression levels and confirming gene expression patterns in different cell types under different conditions, including abiotic and biotic stresses [1,2]
Assessment of Amplification Efficiency and Specificity Based on the sequece of the genes cloned from O. javanica, the Specific primer pairs were designed for the candidate reference genes, with the amplicon lengths ranging from 93 to 192 bp (Table 1)
Cq Values of Candidate Reference Genes To provide an overview of the transcript levels of the nine candidate reference genes, expression levels were determined as quantification cycle (Cq) values (Table S2), and a distribution diagram was drawn (Fig. 1)
Summary
Accuracy, efficiency and reproducibility, Quantitative real-time PCR (qPCR) has become the most prevalent method for quantifying transcript expression levels and confirming gene expression patterns in different cell types under different conditions, including abiotic and biotic stresses [1,2]. To avoid bias from RNA stability, quantity, purity, and enzymatic efficiency in cDNA synthesis and PCR amplification, the data is normalized to one or more of the reference genes [4,5]. Some of the widely used qPCR reference genes in plants and animals include: Actin, 18S ribosomal RNA (18S rRNA), Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Elongation factor-1a (EF-1a), Polyubiquitin (UBQ), and Tubulin (TUB) [8,9,10]. These genes are considered as housekeeping genes with roles in basic cellular processes, and referred as traditional reference genes. Some new reference genes were identified and found to express stably. These reference genes include: genes encoding F-box/kelch-repeat protein (F-box), SAND family protein (SAND), protein phosphatase 2A (PP2A), phosphoenolpyruvate carboxylase-related kinase 1 (PEPKR1), and Tap42-interacting protein of 41 kDa (TIP41) [11,12,13]
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