Evaluation of Angelica decursiva reference genes under various stimuli for RT-qPCR data normalization

Yuedong He,Yanan Gai,Yuan Zhong,Weiqi Wang,Jie Wu,Xiaoqing Xu,Zhenzhen Bao

doi:10.1038/s41598-021-98434-6

Abstract

Angelica decursiva is one of the lending traditional Chinese medicinal plants producing coumarins. Notably, several studies have focused on the biosynthesis and not the RT-qPCR (quantitative real-time reverse transcription polymerase chain reaction) study of coumarins. This RT-qPCR technique has been extensively used to investigate gene expression levels in plants and the selection of reference genes which plays a crucial role in standardizing the data form the RT-qPCR analysis. In our study, 11 candidate reference genes were selected from the existing transcriptome data of Angelica decursiva. Here, four different types of statistical algorithms (geNorm, NormFinder, BestKeeper, and Delta Ct) were used to calculate and evaluate the stability of gene expression under different external treatments. Subsequently, RefFinder analysis was used to determine the geometric average of each candidate gene ranking, and to perform comprehensive index ranking. The obtained results showed that among all the 11 candidate reference genes, SAND family protein (SAND), protein phosphatase 2A gene (PP2A), and polypyrimidine tract-binding protein (PTBP) were the most stable reference genes, where Nuclear cap binding protein 2 (NCBP2), TIP41-like protein (TIP41), and Beta-6-tubulin (TUBA) were the least stable genes. To the best of our knowledge, this work is the first to evaluate the stability of reference genes in the Angelica decursiva which has provided an important foundation on the use of RT-qPCR for an accurate and far-reaching gene expression analysis in this medicinal plant.

Highlights

Angelica decursiva is one of the lending traditional Chinese medicinal plants producing coumarins
There genes are usually used as a reference for either tissues or cells when they are stably expressed in a diversity of experimental conditions, and when changes are detected in the expression level of a target gene
11 candidate reference genes were selected based on the transcriptome data of A. decursiva

Summary

Introduction

Angelica decursiva is one of the lending traditional Chinese medicinal plants producing coumarins. Several studies have focused on the biosynthesis and not the RT-qPCR (quantitative realtime reverse transcription polymerase chain reaction) study of coumarins This RT-qPCR technique has been extensively used to investigate gene expression levels in plants and the selection of reference genes which plays a crucial role in standardizing the data form the RT-qPCR analysis. To the best of our knowledge, this work is the first to evaluate the stability of reference genes in the Angelica decursiva which has provided an important foundation on the use of RT-qPCR for an accurate and far-reaching gene expression analysis in this medicinal plant. Among the most commonly used internal reference genes for RT-qPCR are Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Elongation factor-1α (EF-1α), Tubulin (TUB), β-Actin (ACT), 18S ribosomal RNA protein (18S), Polyubiquitin 10 (UBQ10), etc These housekeeping genes play a significant role in the cell structure maintenance and during primary metabolic activities. Our work provides a basis for further studies on gene expression profiling and the regulation mechanisms of coumarin biosynthesis in A. decursiva under diverse experimental conditions

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Conclusion