Selection of reliable reference genes for RT-qPCR analysis during developmental stages and abiotic stress in Setaria viridis

Celery is one of the most important vegetable crop and its yield and quality is influenced by many environmental factors. Researches on gene expression not only help to unravel the molecular regulatory mechanism but also identify the key genes in the biological response. RT-qPCR is a commonly used technology to quantify the gene expression. Selecting an appropriate reference gene is an effective approach to improve the accuracy of RT-qPCR assay. To our knowledge, the evaluation of reference genes under different treatments in celery has not been reported yet. In this study, the expression stabilities of eight candidate reference genes (ACTIN, eIF-4α, GAPDH, TBP, TUB-A, UBC, TUB-B, and EF-1α) under abiotic stresses (heat, cold, drought, and salt) and hormone treatments (SA, MeJA, GA, and ABA) were detected. The expression stabilities of candidate genes were compared and ranked by geNorm, NormFinder, BestKeeper, ΔCt, and RefFinder programs. The results calculated by different programs were not completely consistent. Considering the comprehensive analysis results, ACTIN was the most stable reference gene and TUB-B showed the worst expression stabilities under the selected abiotic stress and hormone treatments in celery. The reliability of reference genes was further confirmed by the normalization of CAT1 gene under drought stress. This study presented evidences and basis to select the appropriate reference genes under different treatments in celery.

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Selection of reference genes in mouse preimplantation embryos of different ploidies at various developmental stages

Reproduction Abstracts ◽

10.1530/repabs.1.p099 ◽

2014 ◽

Author(s):

Yanli Gu ◽

Xinghui Shen ◽

Dongjie Zhou ◽

Zhendong Wang ◽

Lei Lei

Keyword(s):

Reference Genes ◽

Developmental Stages ◽

Preimplantation Embryos ◽

Selection Of

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Spatio-temporal selection of reference genes in the two congeneric species of Glycyrrhiza

Scientific Reports ◽

10.1038/s41598-020-79298-8 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Yuping Li ◽

Xiaoju Liang ◽

Xuguo Zhou ◽

Yu An ◽

Ming Li ◽

...

Keyword(s):

Gene Expression ◽

Developmental Stage ◽

Reference Genes ◽

Developmental Stages ◽

Individual Factors ◽

Congeneric Species ◽

Spatio Temporal ◽

The Stability ◽

Different Tissues ◽

Selection Of

AbstractGlycyrrhiza, a genus of perennial medicinal herbs, has been traditionally used to treat human diseases, including respiratory disorders. Functional analysis of genes involved in the synthesis, accumulation, and degradation of bioactive compounds in these medicinal plants requires accurate measurement of their expression profiles. Reverse transcription quantitative real-time PCR (RT-qPCR) is a primary tool, which requires stably expressed reference genes to serve as the internal references to normalize the target gene expression. In this study, the stability of 14 candidate reference genes from the two congeneric species G. uralensis and G. inflata, including ACT, CAC, CYP, DNAJ, DREB, EF1, RAN, TIF1, TUB, UBC2, ABCC2, COPS3, CS, R3HDM2, were evaluated across different tissues and throughout various developmental stages. More importantly, we investigated the impact of interactions between tissue and developmental stage on the performance of candidate reference genes. Four algorithms, including geNorm, NormFinder, BestKeeper, and Delta Ct, were used to analyze the expression stability and RefFinder, a comprehensive software, provided the final recommendation. Based on previous research and our preliminary data, we hypothesized that internal references for spatio-temporal gene expression are different from the reference genes suited for individual factors. In G. uralensis, the top three most stable reference genes across different tissues were R3HDM2, CAC and TUB, while CAC, CYP and ABCC2 were most suited for different developmental stages. CAC is the only candidate recommended for both biotic factors, which is reflected in the stability ranking for the spatio (tissue)-temporal (developmental stage) interactions (CAC, R3HDM2 and DNAJ). Similarly, in G. inflata, COPS3, R3HDM2 and DREB were selected for tissues, while RAN, COPS3 and CS were recommended for developmental stages. For the tissue-developmental stage interactions, COPS3, DREB and ABCC2 were the most suited reference genes. In both species, only one of the top three candidates was shared between the individual factors and their interactions, specifically, CAC in G. uralensis and COPS3 in G. inflata, which supports our overarching hypothesis. In summary, spatio-temporal selection of reference genes not only lays the foundation for functional genomics research in Glycyrrhiza, but also facilitates these traditional medicinal herbs to reach/maximize their pharmaceutical potential.

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