Selection and evaluation of reference genes for qRT-PCR analysis in Euscaphis konishii Hayata based on transcriptome data

Abstract

BackgroundQuantitative real-time reverse transcription-polymerase chain reaction has been widely used in gene expression analysis, however, to have reliable and accurate results, reference genes are necessary to normalize gene expression under different experimental conditions. Several reliable reference genes have been reported in plants of Traditional Chinese Medicine, but none have been identified for Euscaphis konishii Hayata.ResultsIn this study, 12 candidate reference genes, including 3 common housekeeping genes and 9 novel genes based on E. konishii Hayata transcriptome data were selected and analyzed in different tissues (root, branch, leaf, capsule and seed), capsule and seed development stages. Expression stability was calculated using geNorm and NormFinder, the minimal number of reference genes required for accurate normalization was calculated by Vn/Vn + 1 using geNorm. EkEEF-5A-1 and EkADF2 were the two most stable reference genes for all samples, while EkGSTU1 and EkGAPDH were the most stable reference genes for tissue samples. For seed development stages, EkGAPDH and EkEEF-5A-1 were the most stable genes, whereas EkGSTU1 and EkGAPDH were identified as the two most stable genes in the capsule development stages. Two reference genes were sufficient to normalize gene expression across all sample sets.ConclusionResults of this study revealed that suitable reference genes should be selected for different experimental samples, and not all the common reference genes are suitable for different tissue samples and/or experimental conditions. In this study, we present the first data of reference genes selection for E. konishii Hayata based on transcriptome data, our data will facilitate further studies in molecular biology and gene function on E. konishii Hayata and other closely related species.Electronic supplementary materialThe online version of this article (10.1186/s13007-018-0311-x) contains supplementary material, which is available to authorized users.

Comprehensive transcriptome analysis of reference genes for fruit development ofEuscaphis konishii

“…In contrast, we confirm that ACT is the most unstable gene in our study. This consequence is consistent with studies in Setaria viridis (Martins et al, 2016) and E. konishii by Liang et al (2018b). UBC has been widely accepted for normalization of gene expression as a reference gene (Yuan, Wan & Yang, 2012).…”

“…This result validates and complements the results of reference gene screening by Liang. (Liang et al, 2018b). The expression of reference genes was related to organ type, developmental stages and external environmental conditions.…”

“…The fruit of E. konishii has various chemical compounds, including flavonoid compounds (Liang et al, 2018a), triterpene compounds (Cheng et al, 2010;Li et al, 2016), and phenolic acid compounds (Huang et al, 2014), which have both anti-inflammatory and anticancer effects according to traditional and modern medical research. Moreover, in order to study the gene regulation mechanisms of medicinal compounds in E. konishii, several reference genes in E. konishii were selected by Liang et al (2019) based on transcriptome data (Liang et al, 2018b). However, the data being studied from Liang et al lack the transcriptome of the fruit at different developmental stages.…”

Selection of the Reference Gene for Expression Normalization in Papaver somniferum L. under Abiotic Stress and Hormone Treatment

“…Therefore, we recommend the top three candidate genes-NCBP2, GAPC2, and TIP41 as reference genes. Interestingly, although a commonly used reference gene, expression of GAPDH in P. somniferum was not stable enough for accurate normalization, but was recommended as one of the best reference genes in Barber [41], E. konishii Hayata [42], and sugarcane under a water deficit environment [27]. This difference demonstrates that no reference gene is universally available, so the selection and validation of suitable reference genes remain indispensable.…”

Screening and Evaluation of Stable Reference Genes for Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) Analysis in Chinese Fir Roots under Water, Phosphorus, and Nitrogen Stresses

“…We also found GAPDH and UBQ as the two most stable genes under drought stress, Actin1 and GAPDH as the two most stable genes under P starvation, and Actin1 and UBC as the two most stable genes under N starvation among the nine candidate reference genes. Thus, not all housekeeping genes are equally valuable to serve as reference genes to normalize the expression level of target genes, as reported previously [55].…”