Stable Internal Reference Genes for Normalizing Real-Time Quantitative PCR in Baphicacanthus cusia under Hormonal Stimuli and UV Irradiation, and in Different Plant Organs

Abstract

Baphicacanthus cusia (Nees) Bremek, the plant source for many kinds of drugs in traditional Chinese medicine, is widely distributed in South China, especially in Fujian. Recent studies about B. cusia mainly focus on its chemical composition and pharmacological effects, but further analysis of the plant's gene functions and expression is required to better understand the synthesis of its effective compounds. Real-time quantitative polymerase chain reaction (RT-qPCR) is a powerful method for gene expression analysis. It is necessary to select a suitable reference gene for expression normalization to ensure the accuracy of RT-qPCR results. Ten candidate reference genes were selected from the transcriptome datasets of B. cusia in this study, and the expression stability was assessed across 60 samples representing different tissues and organs under various conditions, including ultraviolet (UV) irradiation, hormonal stimuli (jasmonic acid methyl ester and abscisic acid), and in different plant organs. By employing different algorithms, such as geNorm, NormFinder, and BestKeeper, which are complementary approaches based on different statistical procedures, 18S rRNA was found to be the most stable gene under UV irradiation and hormonal stimuli, whereas ubiquitin-conjugating enzyme E2 was the best suitable gene for different plant organs. This novel study aimed to screen for suitable reference genes and corresponding primer pairs specifically designed for gene expression studies in B. cusia, in particular for RT-qPCR analyses.

Reference gene selection for qRT-PCR assays inStellera chamaejasmesubjected to abiotic stresses and hormone treatments based on transcriptome datasets

“…18S is a frequently used HKG and is widely used for normalization in qRT-PCR analysis. Wang et al (2017) reported that 18S rRNA was one of the most stably expressed gene under diverse heavy metals stresses in tea plants; Huang et al (2017) also found that 18S rRNA was the most stable gene under UV irradiation and hormonal stimuli in Baphicacanthus cusia . However, our analysis results suggested that 18S was the most unstable RG in all of the experiment groups because of its excessively high expression level.…”

Molecular cloning and metabolomic characterization of the 5-enolpyruvylshikimate-3-phosphate synthase gene from Baphicacanthus cusia

“…Additionally, BcEPSPS in B. cusia is sensitive to glyphosate, which indicates that the BcEPSPS protein is a class I protein. As several glyphosate-resistant mutants of class I EPSPS have been identified, and most of the class I EPSPS mutants display increased glyphosate resistance [21], it is useful to create a glyphosate-resistant mutant of a B. cusia EPSPS using a directed evolution strategy [22].…”

Evaluation of reference genes for reverse transcription quantitative real-time PCR (RT-qPCR) studies in Silene vulgaris considering the method of cDNA preparation

“…18S rRNA is sometimes utilized for expression normalization in angiosperms [ 70 , 71 ], but there are also numerous reports about its expression instability [ 13 , 14 ]. 18S rRNA exhibited variable transcript levels in S .…”