Selection of appropriate reference genes for qPCR in the Chinese mitten crab, Eriocheir sinensis (Decapoda, Varunidae)

Crustaceana ◽  
2017 ◽  
Vol 90 (3) ◽  
pp. 275-296 ◽  
Author(s):  
Shu Huang ◽  
Xiaowen Chen ◽  
Jun Wang ◽  
Jiao Chen ◽  
Wucheng Yue ◽  
...  
Keyword(s):  
Reference Gene ◽  
Reference Genes ◽  
Developmental Stages ◽  
Receptor Gene ◽  
Eriocheir Sinensis ◽  
Chinese Mitten Crab ◽  
Experimental Conditions ◽  
Mitten Crab ◽  
The Stability ◽  
Selection Of

The accuracy of qPCR depends on the stability of the reference gene used for data normalization. However, the stably expressed reference genes in the Chinese mitten crab,Eriocheir sinensis, have not been well identified under different experimental conditions. In this study, the stabilities of the expressions of 10 candidate reference genes were evaluated in different developmental stages, tissues, and moulting stages ofE. sinensis. Our results indicated thatUBEandS27were the most stable reference genes. To validate the suitability of the reference genes, theEcR(ecdysone receptor) gene was analysed among different moulting stages. The results showed that the expression level ofEcRwas elevated using the least stable reference gene,GST, compared with using the three most stable reference genes. Taken together, our results indicate that reference genes should be assessed and selected in accordance with the experimental conditions, and more than one reference gene should be selected.

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

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.

scholarly journals Current Practices for Reference Gene Selection in RT-qPCR of Aspergillus: Outlook and Recommendations for the Future

Genes ◽  
2021 ◽  
Vol 12 (7) ◽  
pp. 960
Author(s):  
Meagan Archer ◽  
Jianping Xu
Keyword(s):  
Gene Expression ◽  
Reference Gene ◽  
Reference Genes ◽  
Gene Selection ◽  
Quantitative Polymerase Chain Reaction ◽  
Specific Method ◽  
Experimental Conditions ◽  
Reference Gene Selection ◽  
Physiological Processes ◽  
The Stability

Aspergillus is a genus of filamentous fungi with vast geographic and ecological distributions. Species within this genus are clinically, agriculturally and biotechnologically relevant, leading to increasing interest in elucidating gene expression dynamics of key metabolic and physiological processes. Reverse-transcription quantitative Polymerase Chain Reaction (RT-qPCR) is a sensitive and specific method of quantifying gene expression. A crucial step for comparing RT-qPCR results between strains and experimental conditions is normalisation to experimentally validated reference gene(s). In this review, we provide a critical analysis of current reference gene selection and validation practices for RT-qPCR gene expression analyses of Aspergillus. Of 90 primary research articles obtained through our PubMed query, 17 experimentally validated the reference gene(s) used. Twenty reference genes were used across the 90 studies, with beta-tubulin being the most used reference gene, followed by actin, 18S rRNA and glyceraldehyde 3-phosphate dehydrogenase. Sixteen of the 90 studies used multiple reference genes for normalisation. Failing to experimentally validate the stability of reference genes can lead to conflicting results, as was the case for four studies. Overall, our review highlights the need to experimentally validate reference genes in RT-qPCR studies of Aspergillus.

Reference gene selection and evaluation for expression analysis using qRT-PCR in Galeruca daurica (Joannis)

2016 ◽  
Vol 107 (3) ◽  
pp. 359-368 ◽  
Author(s):  
Y. Tan ◽  
X.-R. Zhou ◽  
B.-P. Pang
Keyword(s):  
Reference Gene ◽  
Reference Genes ◽  
Target Genes ◽  
Gene Selection ◽  
Developmental Stages ◽  
Pcr Analysis ◽  
Experimental Conditions ◽  
Reference Gene Selection ◽  
Functional Studies ◽  
Qrt Pcr

AbstractQuantitative real-time PCR (qRT-PCR) has been used extensively to analyze gene expression and decipher gene function. To obtain the optimal and stable normalization factors for qRT-PCR, selection and validation of reference genes should be conducted in diverse conditions. In insects, more and more studies confirmed the necessity and importance of reference gene selection. In this study, eight traditionally used reference genes in Galeruca daurica (Joannis) were assessed, using qRT-PCR, for suitability as normalization genes under different experimental conditions using four statistical programs: geNorm, Normfinder, BestKeeper and the comparative ΔCt method. The genes were ranked from the most stable to the least stable using RefFinder. The optimal suite of recommended reference genes was as follows: succinate dehydrogenase (SDHA) and tubulin-alpha (TUB-α) for temperature-treated larvae; ribosomal protein L32, SDHA and glutathione S-transferase were best for all developmental stages; ACT and TUB-α for male and female adults; SDHA and TUB-α were relatively stable and expressed in different tissues, both diapause and non-diapause adults. Reference gene evaluation was validated using expression of two target genes: the P450 CYP6 gene and the heat shock protein gene Hsp70. These results confirm the importance of custom reference gene selection when studies are conducted under diverse experimental conditions. A standardized qRT-PCR analysis procedure for gene functional studies is provided that could be useful in studies on other insect species.

scholarly journals Reference Gene Selection for miRNA qRT-PCR Analysis in Lily

2020 ◽  
Author(s):  
Qian Zhang ◽  
Xue Gao ◽  
Lian-Juan Wang ◽  
Yu-Qian Zhao ◽  
Gui-Xia Jia
Keyword(s):  
Reference Gene ◽  
Stress Responses ◽  
Reference Genes ◽  
Gene Selection ◽  
Developmental Stages ◽  
Pcr Analysis ◽  
Critical Element ◽  
Biotic And Abiotic Stress ◽  
Experimental Conditions ◽  
Qrt Pcr

Abstract Background: The selection of reliable reference genes is a critical element for obtaining accurate gene expression data to assess quantitative real-time polymerase chain reaction (qRT-PCR) performance. It is critical to use suitable reference genes in miRNA qRT-PCR because of short amplification products and large differences in the expression levels of target miRNAs involved in some biological processes. However, in lily, which exhibits a large complex genome but lacks a reference, the available miRNA reference genes for use in qRT-PCR under various treatment conditions are limited, and their reliability has rarely been systematically evaluated.Results: In this study, 8 candidate reference genes, including three classic housekeeping genes and five potential miRNAs from the miRNA library of L. × formolongi, were selected and assessed for expression stability utilizing the BestKeeper, geNorm and Normfinder tools, together with the Delta Ct method, across a diverse set of biotic and abiotic experimental conditions (developmental stages, tissues, heat stress and pathogen defence) to determine the best reference gene(s) for L. × formolongi and L. regale. The final ranking was reordered by using RankAggreg, and the results showed that the novel miRNA PC-3p-67_108977 and the conserved miRNAs miR399a, miR399a and U6 were the most stable genes for L. × formolongi and L. regale, respectively, under all tested experimental conditions. Additionally, PC-3p-67_108977 and U6 were the most suitable genes for qRT-PCR studies in lily.Conclusions: This study provides a comprehensive evaluation of the reliability of reference genes for miRNA studies on development and biotic and abiotic stress responses in different lilies. These results will be beneficial for miRNA identification and functional studies of lilies in the future.

scholarly journals Selection of Suitable Reference Genes Based on Transcriptomic Data in Ginkgo biloba under Different Experimental Conditions

Forests ◽  
2020 ◽  
Vol 11 (11) ◽  
pp. 1217
Author(s):  
Tingting Zhou ◽  
Xiaoming Yang ◽  
Fangfang Fu ◽  
Guibin Wang ◽  
Fuliang Cao
Keyword(s):  
Gene Expression ◽  
Ginkgo Biloba ◽  
Reference Genes ◽  
Developmental Stages ◽  
Deciduous Tree ◽  
Internal Reference ◽  
Experimental Conditions ◽  
Transcriptomic Data ◽  
Suitable Reference ◽  
Selection Of

Ginkgo biloba, a deciduous tree species in the Ginkgo family, has a long history of cultivation in China and is widely used in garden landscapes, medicine, food, and health products. However, few reports have focused on the systematic selection of optimal reference genes based on transcriptomic data in G. biloba. The purpose of our research was to select an internal reference gene suitable for different experimental conditions from thirteen candidate reference genes by the delta cycle threshold (ΔCt) method, geNorm, BestKeeper, NormFinder, and RefFinder programs. The reference genes were used for gene expression analyses of Ginkgo biloba. These results showed that elongation factor 1(EF1) and ubiquitin (UBI) were the best choices for samples of different ginkgo genotypes. The expression of UBI and HAS28 presented the most stable at different developmental stages of ginkgo, and EIF3I and RPII were considered as suitable reference genes in different tissues of ginkgo. For methyl jasmonate (MeJA) treatment, ACA and ACT were identified as the optimal reference genes. For cold stress treatment, RPII and EIF4E were chosen for the gene expression normalizations. HAS28 and GAPDH presented the most stable expression for the heat treatment. To validate the above results, a chalcone synthase gene (GbCHS) in ginkgo was amplified by quantitative real-time polymerase chain reaction (qRT-PCR). Our results provide different suitable reference genes for further gene expression studies in ginkgo.

scholarly journals Selection and Validation of Reference Genes Desirable for Gene Expression Analysis by qRT-PCR on Seed Germination of Castanea henryi

2021 ◽  
Author(s):  
Bin Liu ◽  
Yuting Jiang ◽  
Ruqiang Lin ◽  
Yuanfang Xiong ◽  
Shuzhen Jiang ◽  
...  
Keyword(s):  
Seed Germination ◽  
Candidate Genes ◽  
Reference Gene ◽  
Reference Genes ◽  
Stable Gene ◽  
Internal Reference ◽  
Seed Biology ◽  
Unstable Gene ◽  
The Stability ◽  
Selection Of

AbstractSeed germination is the beginning of the plant’s life cycle, and seed biology is one of the most extensively researched areas in plant physiology, however, Castanea henryi as an important seed plant, the stable internal reference gene during germination is not clear. In this study, seven candidate genes (TUA, TUB, TIF, UBC, RPL21, RPL30, RPL34) were screened out from transcriptome data, we analyzed the expression of seven candidate reference genes in C. henryi at different germination stages with RT–qPCR, and using common algorithms including NormFinder, geNorm and BestKeeper to evaluate the candidate genes stability. The results showed that RPL34 and RPL30 were selected as the most stable genes by NormFinder; TIF was the most stable gene identified by BestKeeper; RPL34 and RPL21 were the most stable genes ranked by geNorm, and TUB was the most unstable gene identified by all of the three software. The RPL34 gene was used as the reference gene, to detected the expression trend of two starch synthetase genes SS1 and SS2 during germination by RT–qPCR, the results of RT–qPCR and transcriptome sequencing were basically consistent, which verified the stability of RPL34 candidate gene. Our result is not only showed functional genes for germination of C. henryi seeds and provide useful guidelines for the selection of reliable reference genes for the normalization of RT– qPCR data for germination of seed plants.

scholarly journals Selection and Validation of Suitable Reference Genes for RT-qPCR Analysis in Bursaphelenchus Xylophilus 

2021 ◽  
Author(s):  
Yaning Liu ◽  
Jiao Zhou ◽  
Hongxia Zhang ◽  
Faheem Ahmad ◽  
Jianting Fan ◽  
...  
Keyword(s):  
Gene Expression ◽  
Reference Genes ◽  
Gene Selection ◽  
Developmental Stages ◽  
Genetic Research ◽  
Bursaphelenchus Xylophilus ◽  
Stable Gene ◽  
Pinewood Nematode ◽  
Experimental Conditions ◽  
The Stability

Abstract Quantitative reverse transcription polymerase chain reaction (RT-qPCR) is a powerful technique for studying gene expression, and it is widely used in molecular biology and genetic research. The key to quantitative accuracy depends on the stability of the reference genes used for data normalization under different experimental conditions. The pinewood nematode (PWN; Bursaphelenchus xylophilus) is the causal agent of a devastating disease, the pine wood disease (PWD), demanding extensive and prompt research to understand the molecular mechanism of PWD, but the identification of reference PWN genes for standardized RT-qPCR has not been reported yet. In this study, we have analyzed eight candidate reference genes of PWN under different temperature conditions and at different developmental stages. Delta Ct method, GeNorm, NormFinder, BestKeeper and RefFinder algorithms were used to evaluate the expression stability of these genes. 18SrRNA and HIS were selected for gene expression research under temperature treatments, while EF1γ and 18SrRNA for gene expression studies across different developmental stages. In general, the results indicate that 18SrRNA is the most stable gene for RT-qPCR under different conditions. Finally, we use arginine kinase gene (AK) in different temperature and heat shock protein 90 (HSP90) in different developmental stages to confirm the stability of expression. The systematic analysis of qRT-PCR reference gene selection of B. xylophilus will be helpful for future functional analysis and exploration of genetic resources of B. xylophilus.

scholarly journals Evaluation of Reference Genes in Glenea cantor (Fabricius) by Using qRT-PCR

Genes ◽  
2021 ◽  
Vol 12 (12) ◽  
pp. 1984
Author(s):  
Ran-Ran Su ◽  
Zhong-Yan Huang ◽  
Chao-Wei Qin ◽  
Xia-Lin Zheng ◽  
Wen Lu ◽  
...  
Keyword(s):  
Reference Genes ◽  
Developmental Stages ◽  
Optimal Number ◽  
Experimental Conditions ◽  
Qrt Pcr ◽  
Adult Tissues ◽  
Biological Studies ◽  
Main Host ◽  
The Stability ◽  
Suitable Reference

Kapok is the main host of Glenea cantor (Fabricius), which causes serious damage and is difficult to control. In severe cases, it often causes the kapok trees to die continuously, which seriously affects the results of urban landscaping. To provide reference for the functional research on related genes in G. cantor, we screened the stable expression of candidate reference genes at different developmental stages (i.e., eggs, larvae, pupae, and adults), in various adult tissues (i.e., head, thorax, abdomen, feet, antennae, and wings), and sexes (i.e., male pupae, female pupae, male adults, and female adults). In this study, 12 candidate reference genes (i.e., ACTINLIKE, ACTININ, TUB, RPL36, RPL32, RPS20, TBP, GAPDH, 18S rRNA, EF1A1, EF1A2, and UBQ) were evaluated using different adult tissues, developmental stages, and sexes. RefFinder, geNorm, NormFinder, and BestKeeper were used to evaluate and comprehensively analyze the stability of the expression of the candidate reference genes. The results show that RPL32 and EF1A1 were the most suitable reference genes in the different adult tissues, and RPL36 and EF1A1 were best at the different developmental stages. RPL36 and EF1A2 were the best fit for the qRT-PCR reference genes in the different sexes, while RPL36 and EF1A1 were the most appropriate qRT-PCR reference genes in all samples. Results from geNorm showed that the optimal number of reference genes was two. We also surveyed the expression of cellulase at the different developmental stages and in the different adult tissues. Results further verified the reliability of the reference genes, and confirmed the best reference genes under the different experimental conditions. This study provides a useful tool for molecular biological studies on G. cantor.

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