Identification and Selection of Reference Genes for Quantitative Transcript Analysis in Corydalis yanhusuo

Abstract

Corydalis yanhusuo is a medicinal plant frequently used in traditional Chinese medicine, which has effective medical effects in many aspects. Real-time polymerase chain reaction (RT-PCR) has been one of the most widely used methods in biosynthesis research due to its high sensitivity and quantitative properties in gene expression analysis. To obtain accurate normalization, reference genes are often selected in advance; however, no reference genes are available in C. yanhusuo. Herein, 12 reference gene candidates, named cyclophilin 2 (CYP2), elongation factor 1-α (EF1-α), protein phosphatase 2 (PP2A), SAND protein family (SAND), polypyrimidine tract-binding protein (PTBP), TIP41-like protein (TIP41), lyceraldehyde-3-phosphate hydrogenase (GAPDH), ubiquitin-conjugating enzyme 9 (UBC9), cyclophilin 1 (CYP1), tubulin beta (TUBA), thioredoxin (YLS8), and polyubiquitin 10 (UBQ10), were selected for stability analysis. After being treated with hormone, UV, salt, metal, oxidative, drought, cold (4 °C), and hot stresses (40 °C), the qRT-PCR data of the selected genes was analyzed with NormFinder, geNorm, and BestKeeper. The result indicated that GAPDH, SNAD, and PP2A were the top three most stable reference genes under most treatments. This study selected and validated reliable reference genes in C. yanhusuo under various environmental conditions, which can provide great help for future research on gene expression normalization in C. yanhusuo.

Integration of full-length transcriptomics and targeted metabolomics to identify benzylisoquinoline alkaloid biosynthetic genes in Corydalis yanhusuo

“…The primer pairs used are shown in Table S5. The relative expression levels of the target genes were calculated using the 2 −ΔΔCT approach with GAPDH as the reference gene 63 . Each gene was analyzed in three biological replicates and three technical replicates.…”

Selection of Suitable Reference Genes for Gene Expression Normalization Studies in Dendrobium huoshanense

“…Unexpectedly, although GAPDH is a commonly used reference gene, its expression in D. huoshanense showed fairly poor expression stability under all experimental conditions in our research, which meant that it could not be used as a stable reference gene for qPCR analysis. However, it was regarded as the best reference gene in D. catenatum [ 57 ], Diabrotica undecimpunctata [ 58 ], and Corydalis yanhusuo [ 59 ]. Similarly, although TIP41 is not suitable as an internal reference gene in this study, its expression presented high stability in Momordica charantia [ 60 ].…”

Selection of a reference gene for studies on lipid‐related aquatic adaptations of toothed whales (Grampus griseus)

“…Considering all of these aspects, we determined candidate genes with the lowest CV percentage. Reference genes for qPCR can be identified through high‐throughput transcriptomic data by next‐generation sequencing (Bao et al, 2020; Gao et al, 2018; Pombo et al, 2018). All of our selected candidate reference genes exhibited a high level of expression by more than 500 FPKM values, and, according to multiple sources, their functions include regulatory and maintenance functions, metabolic processes, and catalysis in cells.…”