Assessment of reference genes for real-time quantitative PCR normalization in Ilex paraguariensis leaves during drought

Abstract

Reverse transcription of RNA followed by real-time quantitative PCR (qPCR) is to date the most reliable method for gene expression studies. However, to control the errors introduced along the numerous experimental procedures, it requires a normalization using internal reference genes with stable expression. To address this issue, nine candidate reference genes were investigated in Ilex paraguariensis leaves subjected to water stress. To facilitate the selection, we analysed the real-time qPCR data with three different software programs. The obtained results support the conclusion that RNA polymerase associated protein rtf1 homolog (RTF) combined with any of the following pairs is the most suitable triad of genes to compute a normalization factor: elongation factor 1-alpha + tubulin alpha chain (EF1a + α-Tub), actin + cyclophilin 38 (ACT + CYP38), or cyclophilin 38 + vacuolar protein sorting-associated protein 18 homologs (CYP38 + VPS). Our analysis constitutes the first in-depth study to identify the appropriate reference genes for the quantification of transcription in Ilex paraguariensis leaves during drought and provides essential information for further gene expression studies in this tree species.

Identification of Suitable Reference Genes for RT-qPCR Assays in Liriodendron chinense (Hemsl.) Sarg

“…In contrast, UBQ1 and AGD11 were unstably expressed genes in L. chinense (Table 5). As a member of the UBQ family, UBQ1 did not have good expression stability in our study, and it also showed an unstable expression pattern in lettuce (Lactuca sativa) [8] and in leaves of Ilex paraguariensis under drought treatment [46]. However, UBQ1 had better expression stability in the parasitic life cycle of Striga hermonthica [10].…”

Reference gene selections for real time quantitative PCR analysis of gene expression in different oat tissues and under salt stress

“…The reliability of the data generated in such studies is critically dependent on the identification of suitable reference genes. Such internal controls have been identified and validated for a variety of plant species (Paolacci et al 2009, Manoli et al 2012, Saha et al 2014, Acevedo et al 2018. However, to our knowledge, no systematic evaluation of appropriate reference genes in cultivated oat has yet been reported.…”

Reference gene selection in multiple Passiflora edulis tissues and under cold stress for RT- qPCR analysis

“…EF1 is stably expressed in not only P. edulis, but also other plants. For example, EF1 was used as an internal control gene in Ilex paraguariensis leaves and potato (Solanum tuberosum L.) under abiotic stress [43,44]. To our knowledge, the expression level of reference genes may change with changes in species, experimental conditions, and tissue types.…”

“…For example, although EF1 was suitable for data normalization in P. edulis, potato under abiotic stress, drought-treated leaves of I. paraguariensis, and Betula luminifera, this gene was unsuitable for data normalization in Camellia sinensis under metal stress and in leaves of Camellia sinensis [38,[43][44][45][46]. Overall, Ts and EF1 have the highest expression stability, but if we classify all samples into different subsets, there may be other genes in each subset with higher expression stability than Ts and EF1.…”