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

Yi, Shanyong; Lu, Haibo; Tian, Canrong; Xu, Tao; Song, Cheng; Wang, Wei; Wei, Peipei; Gu, Fei; Liu, Dong; Cai, Yongping; Han, Bangxing

doi:10.3390/genes13081486

Cited by 7 publications

(6 citation statements)

References 60 publications

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“…In addition, we compared the expression levels of AtACT2 (Arabidopsis) with those of ACT7 and the stable reference genes RPL27 and RPS15 (C. burmnnii) under the Nacl-treated samples (Figure S3), showing that the stability of At-ACT2 was relatively lower. Based on the previous research, TATA-box, as the first promoter found in eukaryotes, was more suitable for q−PCR analysis in a variety of species, such as in Monomorium pharaonic [1], Gleditsia microphylla [56] and Dendrobium huoshanense [57], but in our study it was not the optimum reference gene for some experimental conditions. In addition, eIF-5A was just the best reference gene in different borneol clones and EF1α was the optimum gene for studying different tissues.…”

Section: Discussionmentioning

confidence: 72%

Reference Genes Selection and Validation for Cinnamomum burmanni by Real-Time Quantitative Polymerase Chain Reaction

Shi,

Cai,

Yao

et al. 2024

IJMS

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In recent years, the field of biology has witnessed a surge of interest in genomics research due to the advancements in biotechnology. Gene expression pattern analysis plays a crucial role in this research, as it enables us to understand the regulatory mechanism of gene expression and the associated biological processes. Real-time quantitative polymerase chain reaction (q-PCR) is an efficient method to analyze the gene expression patterns, for which accuracy relies on the standardized analysis of reference genes. However, numerous studies have shown that no reference gene is universal in all conditions, so screening a suitable reference gene under certain conditions is of great importance. Cinnamomum burmannii (C. burmannii) is rich in volatile components and has high medicinal and economic value. However, knowledge of the screening of reference genes for the gene expression analysis of C. burmannii is insufficient. Aiming at this problem, we evaluated and screened the reference genes in C. burmannii under different experimental conditions, including different abiotic stresses (Cold-treated, PEG-treated and Nacl-treated), different tissues, leaves at different developmental stages and different chemical types. In this study, different algorithms (∆Ct, geNorm, NormFinder and BestKeeper) were used to evaluate the stability of the candidate reference genes, and RefFinder further merged the output data to screen out the optimum reference gene under various experimental conditions in C. burmannii. The results showed that the optimal reference gene number for gene standardization was 2 under different experimental conditions. RPL27|RPS15 was the most suitable combination under the Nacl-treated and PEG-treated samples. RPL27|APT was the optimum combination under the Cold-treated samples. The optimal combinations of other samples were EF1α|ACT7 for different tissues, eIF-5A|Gllα for different borneol clones in C. burmannii, RPS15|ACT7 for leaves at different developmental stages and RPS15|TATA for all samples. Additionally, two terpenoid synthesis-related genes (CbWRKY4 and CbDXS2) were standardized to verify the feasibility of the selected reference genes under different experimental conditions. This study will be helpful for the subsequent molecular genetic mechanism study of C. burmannii.

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Section: Discussionmentioning

confidence: 72%

Reference Genes Selection and Validation for Cinnamomum burmanni by Real-Time Quantitative Polymerase Chain Reaction

Shi,

Cai,

Yao

et al. 2024

IJMS

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“…These results agreed with previous studies conducted on Momordica charantia [22] and Psoralea corylifolia [21], where TIP41 also demonstrated high stability. However, in studies on Angelica decursiva [17] and Dendrobium huoshanense [44], TIP41 was not identified as the most stable gene, further highlighting the absence of a universal reference gene that is stably expressed across different conditions or organisms. Hence, it is essential to verify presumed reference genes before each RT-qPCR analysis to ensure their suitability for specific experimental conditions.…”

Section: Discussionmentioning

confidence: 97%

Selection and Verification of Standardized Reference Genes of Angelica dahurica under Various Abiotic Stresses by Real-Time Quantitative PCR

Zhang,

He,

Zhou

et al. 2024

Genes

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In traditional Chinese medicine, Angelica dahurica is a valuable herb with numerous therapeutic applications for a range of ailments. There have not yet been any articles on the methodical assessment and choice of the best reference genes for A. dahurica gene expression studies. Real-time quantitative PCR (RT-qPCR) is widely employed as the predominant method for investigating gene expression. In order to ensure the precise determination of target gene expression outcomes in RT-qPCR analysis, it is imperative to employ stable reference genes. In this study, a total of 11 candidate reference genes including SAND family protein (SAND), polypyrimidine tract-binding protein (PTBP), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin (ACT), TIP41-like protein (TIP41), cyclophilin 2 (CYP2), elongation factor 1 α (EF1α), ubiquitin-protein ligase 9 (UBC9), tubulin β-6 (TUB6), thioredoxin-like protein YLS8 (YLS8), and tubulin-α (TUBA) were selected from the transcriptome of A. dahurica. Subsequently, three statistical algorithms (geNorm, NormFinder, and BestKeeper) were employed to assess the stability of their expression patterns across seven distinct stimulus treatments. The outcomes obtained from these analyses were subsequently amalgamated into a comprehensive ranking using RefFinder. Additionally, one target gene, phenylalanine ammonia-lyase (PAL), was used to confirm the effectiveness of the selected reference genes. According to the findings of this study, the two most stable reference genes for normalizing the expression of genes in A. dahurica are TIP41 and UBC9. Overall, our research has determined the appropriate reference genes for RT-qPCR in A. dahurica and provides a crucial foundation for gene screening and identifying genes associated with the biosynthesis of active ingredients in A. dahurica.

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“…These primers were synthesized by Sangon Biotech (Sangon, Shanghai, China). The 2 −ΔΔCT method was used for relative quantitative analysis of the data, and the internal reference gene TATA box binding protein-like ( TBP ) [ 35 ] and SYBR Premix Ex Taq II (Novoprotein, Shanghai, China) were employed to validate the DEG expression results. Three biological and three technical replicates of each reaction were analyzed on a LightCycler ® 480 instrument (Roche, Switzerland) with a first step of 95 °C for 5 min for pre-denaturation, followed by 40 cycles of 95 °C for 10 s for denaturation, and 60 °C for 30 s for annealing/extension [ 35 ].…”

Section: Methodsmentioning

confidence: 99%

Transcriptomic and Metabolomic Analyses Reveal Differences in Flavonoid Pathway Gene Expression Profiles between Two Dendrobium Varieties during Vernalization

Shu

Shi

Zhang

et al. 2023

IJMS

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Dendrobium (Orchidaceae, Epidendoideae) plants have flowers with a wide variety of colors that persist for a long period throughout the year. The yellow coloration of Dendrobium flowers is mainly determined by the flavonol pathway and the flavone pathway, but the relevant biosynthesis mechanisms during vernalization remain unclear. To explore the similarities and differences in flavonoid biosynthesis in different tissues during vernalization, we selected two species of Dendrobium for a flower color study: Dendrobium capillipes Rchb (which has yellow flowers) and Dendrobium nobile Lindl (which has white flowers). We collected a total of 36 samples from six tissue types and both Dendrobium species during vernalization and subjected the samples to metabolic profiling and transcriptome sequencing. A total of 31,504 differentially expressed genes (DEGs) were identified between different tissues of the two Dendrobium species by transcriptomic analysis. However, many differentially accumulated metabolites (DAMs) and DEGs were enriched not only in the general pathway of “flavonoid biosynthesis” but also in multiple subpathways of “flavone and flavonol biosynthesis”. According to a combined transcriptome and metabolome analysis, Putrescine hydroxycinnamoyl transferase 1 (LOC110093422) may be the main gene responsible for the differences in flavonoid accumulation during vernalization, which is closely associated with yellow flowers. Taken together, the results of our study preliminarily revealed the metabolites responsible for and the key genes regulating flavonoid biosynthesis during vernalization. These results provide a basis for the further study of the molecular mechanism of flavonoid synthesis during vernalization.

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

Cited by 7 publications

References 60 publications

Reference Genes Selection and Validation for Cinnamomum burmanni by Real-Time Quantitative Polymerase Chain Reaction

Reference Genes Selection and Validation for Cinnamomum burmanni by Real-Time Quantitative Polymerase Chain Reaction

Selection and Verification of Standardized Reference Genes of Angelica dahurica under Various Abiotic Stresses by Real-Time Quantitative PCR

Transcriptomic and Metabolomic Analyses Reveal Differences in Flavonoid Pathway Gene Expression Profiles between Two Dendrobium Varieties during Vernalization

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