Selection and Validation of Appropriate Reference Genes for qRT-PCR Analysis in Isatis indigotica Fort.

Abstract

Due to its sensitivity and specificity, real-time quantitative PCR (qRT-PCR) is a popular technique for investigating gene expression levels in plants. Based on the Minimum Information for Publication of Real-Time Quantitative PCR Experiments (MIQE) guidelines, it is necessary to select and validate putative appropriate reference genes for qRT-PCR normalization. In the current study, three algorithms, geNorm, NormFinder, and BestKeeper, were applied to assess the expression stability of 10 candidate reference genes across five different tissues and three different abiotic stresses in Isatis indigotica Fort. Additionally, the IiYUC6 gene associated with IAA biosynthesis was applied to validate the candidate reference genes. The analysis results of the geNorm, NormFinder, and BestKeeper algorithms indicated certain differences for the different sample sets and different experiment conditions. Considering all of the algorithms, PP2A-4 and TUB4 were recommended as the most stable reference genes for total and different tissue samples, respectively. Moreover, RPL15 and PP2A-4 were considered to be the most suitable reference genes for abiotic stress treatments. The obtained experimental results might contribute to improved accuracy and credibility for the expression levels of target genes by qRT-PCR normalization in I. indigotica.

Reference genes for RT-qPCR normalisation in different tissues, developmental stages and stress conditions of Hypericum perforatum

“…The reliability of qRT-PCR data will be greatly improved by the inclusion of a reference gene with a transcription level that is invariable across different experimental conditions [8]. In many published studies, some common reference genes especially the housekeeping genes have been verified in many species, such as in Arabidopsis thaliana [46,54], Poa pratensis [54], Salvia miltiorrhiza [55], Cucumis sativus [56], Gentiana macrophylla [37], and Isatis indigotica [57]. But selecting appropriate reference genes for H. perforatums in different experimental conditions have not yet been conducted, which might be due to the fact that genomic information for H. perforatums is very limited in NCBI.…”

“…Homologous genes are widely used as reference genes for gene expression analysis. For example, ACT6, ACT8, and ACT7 were selected as internal controls for stress treatments in Fortunella crassifolia [33]; UBC19, UBC22 , and UBC29 were selected as reference genes in the context of the relevant experimental conditions in Isatis indigotica [57]; and EF1A2a, EF1A2b , and EF1A1a1 were the best reference genes under all tested conditions in Glycine max [68]. Nevertheless, as can be seen in our results, the three homologous genes ( ACT2, ACT3 and ACT7 ) exhibited totally different expression levels when used for the normalization of qRT-PCR, especially ACT2 and ACT3 (Table 2, Table 3, Table 4 and Fig.…”

“…3). Therefore the paralogous genes have similar stuctures, but their expression levels are entirely differnet in gene expression quantification [57].…”

“…Three mathematical programs, geNorm [31], NormFinder [32] and BestKeeper [33], were applied to analyse the raw Ct values and to figure out the relatively stable expressed genes. All of the raw Ct values need to be converted to ΔCt (ΔCt = each corresponding average Ct value - mimimum Ct value) in NormFinder and geNorm algorithms, while there is no need in BestKeeper [34]. The selected reference genes were detected by using a target gene HpHYP1 belonging to the Pathogenesis related class-10 (PR-10) family [35,36].…”

Curcumin attenuates endothelial cell fibrosis through inhibiting endothelial–interstitial transformation

“…The specific qRT-PCR experimental methods were performed as previously described. 50 The sequences of the primers used are listed in Table 2.…”