scholarly journals Selection and evaluation of potential reference genes for gene expression analysis in Avena fatua Linn

2018 ◽  
Vol 55 (No. 1) ◽  
pp. 61-71 ◽  
Author(s):  
Junjie Liu ◽  
Peng Li ◽  
Liuyang Lu ◽  
Lanfen Xie ◽  
Xiling Chen ◽  
...  
Keyword(s):  
Reference Genes ◽  
Developmental Stages ◽  
Elongation Factor ◽  
Avena Fatua ◽  
28S Rrna ◽  
Experimental Conditions ◽  
Alpha Tubulin ◽  
Herbicide Treatments ◽  
Tata Box Binding Protein ◽  
Suitable Reference

Eight commonly used candidate reference genes, 18S ribosomal RNA (rRNA) (18S), 28S rRNA (28S), actin (ACT), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), elongation factor 1 alpha (EF1α), ribosomal protein L7 (RPL7), Alpha-tubulin (α-TUB), and TATA box binding protein-associated factor (TBP), were evaluated under various experimental conditions to assess their suitability in different developmental stages, tissues and herbicide treatments in Avena fatua. The results indicated the most suitable reference genes for the different experimental conditions. For developmental stages, 28S and EF1α were the optimal reference genes, both EF1α and 28S were suitable for experiments of different tissues, whereas for herbicide treatments, GAPDH and ACT were suitable for normalizations of expression data. In addition, GAPDH and EF1α were the suitable reference genes.

scholarly journals Reference Gene Selection for qPCR Normalization ofKosteletzkya virginicaunder Salt Stress

2015 ◽  
Vol 2015 ◽  
pp. 1-8 ◽  
Author(s):  
Xiaoli Tang ◽  
Hongyan Wang ◽  
Chuyang Shao ◽  
Hongbo Shao
Keyword(s):  
Gene Expression ◽  
Reference Gene ◽  
Reference Genes ◽  
Gene Selection ◽  
Expression Profiles ◽  
Elongation Factor ◽  
Gene Expression Profiles ◽  
Stable Reference Gene ◽  
Experimental Conditions ◽  
Suitable Reference

Kosteletzkya virginica(L.) is a newly introduced perennial halophytic plant. Presently, reverse transcription quantitative real-time PCR (qPCR) is regarded as the best choice for analyzing gene expression and its accuracy mainly depends on the reference genes which are used for gene expression normalization. In this study, we employed qPCR to select the most stable reference gene inK. virginicawhich showed stable expression profiles under our experimental conditions. The candidate reference genes were 18S ribosomal RNA (18SrRNA),β-actin (ACT),α-tubulin (TUA), and elongation factor (EF). We tracked the gene expression profiles of the candidate genes and analyzed their stabilities through BestKeeper, geNorm, and NormFinder software programs. The results of the three programs were identical and18SrRNAwas assessed to be the most stable reference gene in this study. However,TUAwas identified to be the most unstable. Our study proved again that the traditional reference genes indeed displayed a certain degree of variations under given experimental conditions. Importantly, our research also provides guidance for selecting most suitable reference genes and lays the foundation for further studies inK. virginica.

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 of Reference Genes for Normalization of Gene Expression in Thermobia domestica (Insecta: Zygentoma: Lepismatidae)

Genes ◽  
2020 ◽  
Vol 12 (1) ◽  
pp. 21
Author(s):  
Yu Bai ◽  
Ya-Nan Lv ◽  
Mei Zeng ◽  
Pei-Yao Jia ◽  
Hu-Na Lu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Reference Genes ◽  
Developmental Stages ◽  
Gene Expression Pattern ◽  
Elongation Factor ◽  
Body Parts ◽  
Internal Reference ◽  
Experimental Conditions ◽  
Thermobia Domestica ◽  
Insect Metamorphosis

Zygentoma occupies a key evolutionary position for understanding the evolution of insect metamorphosis but has received little attention in terms of genetic analysis. To develop functional genomic studies in this insect, we evaluated five candidate internal reference genes for quantitative RT-PCR (qPCR) studies from Thermobia domestica, a representative species of Zygentoma, including Actin 5C (Actin5C), Elongation factor-1 alpha (EF1A), Ribosome protein S26 (RPS26), Ribosome protein L32 (RPL32), and Superoxide dismutase 2 (SOD2), at different developmental stages, in various body parts, and under dsRNA microinjection and starvation stresses, using four algorithms (delta Ct, geNorm, NormFinder and BestKeeper) and a comparative algorithm (RefFinder). Specific suitable reference genes were recommended across specific experimental conditions, and the combination of RPS26 and RPL32 was appropriate for all tested samples. Employing our selected reference gene combination, we investigated the gene expression pattern of Myoglianin (Myo), a crucial gene-regulating insect metamorphosis, in ametabolous T. domestica, and demonstrated the efficiency of RNA interference (RNAi) in firebrat nymphs. This study provides a basis for reliable quantitative studies of genes and greatly benefits evolutionary and functional genomics studies in Zygentoma.

scholarly journals Identification and Evaluation of Suitable Reference Genes for RT-qPCR Analysis in Hippodamia variegata (Coleoptera: Coccinellidae) Under Different Biotic and Abiotic Conditions

2021 ◽  
Vol 12 ◽  
Author(s):  
Jiaoxin Xie ◽  
Tinghui Liu ◽  
Adel Khashaveh ◽  
Chaoqun Yi ◽  
Xiaoxu Liu ◽  
...  
Keyword(s):  
Reference Genes ◽  
Target Genes ◽  
Developmental Stages ◽  
Quantitative Polymerase Chain Reaction ◽  
Experimental Conditions ◽  
Functional Studies ◽  
Qpcr Analysis ◽  
Hippodamia Variegata ◽  
Convenient Technique ◽  
Suitable Reference

Reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) is an accurate and convenient technique for quantifying expression levels of the target genes. Selection of the appropriate reference gene is of the vital importance for RT-qPCR analysis. Hippodamia variegata is one of the most important predatory natural enemies of aphids. Recently, transcriptome and genome sequencings of H. variegata facilitate the gene functional studies. However, there has been rare investigation on the detection of stably expressed reference genes in H. variegata. In the current study, by using five analytical tools (Delta Ct, geNorm, NormFinder, BestKeeper, and RefFinder), eight candidate reference genes, namely, Actin, EF1α, RPL7, RPL18, RPS23, Tubulin-α, Tubulin-β, and TufA, were evaluated under four experimental conditions including developmental stages, tissues, temperatures, and diets. As a result, a specific set of reference genes were recommended for each experimental condition. These findings will help to improve the accuracy and reliability of RT-qPCR data, and lay a foundation for further exploration on the gene function of H. variegata.

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.

scholarly journals Selection of suitable reference genes for gene expression studies in myxosporean (Myxozoa, Cnidaria) parasites

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Anush Kosakyan ◽  
Gema Alama-Bermejo ◽  
Pavla Bartošová-Sojková ◽  
Ana Born-Torrijos ◽  
Radek Šíma ◽  
...  
Keyword(s):  
Gene Expression ◽  
Rna Polymerase Ii ◽  
Ribosomal Rna ◽  
Reference Genes ◽  
Developmental Stages ◽  
Elongation Factor ◽  
Eukaryotic Translation ◽  
Expression Studies ◽  
Gene Expression Studies ◽  
Suitable Reference

Abstract Myxozoans (Cnidaria: Myxozoa) are an extremely diversified group of endoparasites some of which are causative agents of serious diseases in fish. New methods involving gene expression studies have emerged over the last years to better understand and control myxozoan diseases. Quantitative RT-PCR is the most extensively used approach for gene expression studies. However, the accuracy of the results depends on the normalization of the data to reference genes. We studied the expression of eight commonly used reference genes, adenosylhomocysteinase (AHC1), beta actin (ACTB), eukaryotic translation elongation factor 2 (EF2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), hypoxanthine-guanine phosphoribosyltransferase 1 (HPRT1), DNA-directed RNA polymerase II (RPB2), 18S ribosomal RNA (18S), 28S ribosomal RNA (28S) across different developmental stages of three myxozoan species, Sphaerospora molnari, Myxobolus cerebralis and Ceratonova shasta, representing the three major myxozoan linages from the largest class Myxosporea. The stable reference genes were identified using four algorithms: geNorm, NormFinder, Bestkeeper and ΔCq method. Additionally, we analyzed transcriptomic data from S. molnari proliferative and spore-forming stages to compare the relative amount of expressed transcripts with the most stable reference genes suggested by RT-qPCR. Our results revealed that GAPDH and EF2 are the most uniformly expressed genes across the different developmental stages of the studied myxozoan species.

scholarly journals Selection and Validation of Reference Genes for RT-qPCR Normalization in Bradysia odoriphaga (Diptera: Sciaridae) Under Insecticides Stress

2022 ◽  
Vol 12 ◽  
Author(s):  
Haiyan Fu ◽  
Tubiao Huang ◽  
Cheng Yin ◽  
Zhenhua Xu ◽  
Chao Li ◽  
...  
Keyword(s):  
Gene Expression ◽  
Ribosomal Protein ◽  
Reference Genes ◽  
Gene Selection ◽  
Developmental Stages ◽  
Resistance Mechanism ◽  
Elongation Factor ◽  
Expression Stability ◽  
Experimental Conditions ◽  
Gene Expression Studies

Bradysia odoriphaga (Diptera: Sciaridae) is the most serious root maggot pest which causes substantial damage to the Chinese chive. Organophosphate (OP) and neonicotinoid insecticides are widely used chemical pesticides and play important roles in controlling B. odoriphaga. However, a strong selection pressure following repeated pesticide applications has led to the development of resistant populations of this insect. To understand the insecticide resistance mechanism in B. odoriphaga, gene expression analysis might be required. Appropriate reference gene selection is a critical prerequisite for gene expression studies, as the expression stability of reference genes can be affected by experimental conditions, resulting in biased or erroneous results. The present study shows the expression profile of nine commonly used reference genes [elongation factor 1α (EF-1α), actin2 (ACT), elongation factor 2α (EF-2α), glucose-6-phosphate dehydrogenase (G6PDH), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ribosomal protein L10 (RPL10), ribosomal protein S3 (RPS3), ubiquitin-conjugating enzyme (UBC), and α-tubulin (TUB)] was systematically analyzed under insecticide stress. Moreover, we also evaluated their expression stability in other experimental conditions, including developmental stages, sexes, and tissues. Five programs (NormFinder, geNorm, BestKeeper, RefFinder, and ΔCt) were used to validate the suitability of candidate reference genes. The results revealed that the most appropriate sets of reference genes were RPL10 and ACT across phoxim; ACT and TUB across chlorpyrifos and chlorfluazuron; EF1α and TUB across imidacloprid; EF1α and EF2α across developmental stages; RPL10 and TUB across larvae; EF1α and ACT across tissues, and ACT and G6PDH across sex. These results will facilitate the standardization of RT-qPCR and contribute to further research on B. odoriphaga gene function under insecticides stress.

scholarly journals Selection and Evaluation of Appropriate Reference Genes for RT-qPCR Normalization of Volvariella volvacea Gene Expression under Different Conditions

2018 ◽  
Vol 2018 ◽  
pp. 1-12 ◽  
Author(s):  
Jiang Qian ◽  
Yingnv Gao ◽  
Ying Wáng ◽  
Yingying Wu ◽  
Ying Wāng ◽  
...  
Keyword(s):  
Ribosomal Rna ◽  
Reference Genes ◽  
Developmental Stages ◽  
Volvariella Volvacea ◽  
Experimental Conditions ◽  
Alpha Tubulin ◽  
Wide Range ◽  
Straw Mushroom ◽  
Potential Factors ◽  
Qpcr Normalization

Volvariella volvacea (V. volvacea), commonly referred to as Chinese (paddy straw) mushroom, is a basidiomycete with a protein-rich volva and pileus. Selecting appropriate reference genes is a crucial step in the normalization of quantitative real-time PCR data. Therefore, 12 candidate reference genes were selected from the V. volvacea transcriptome based on previous studies and then BestKeeper, geNorm, and NormFinder were used to identify reference genes stably expressed during different developmental stages and conditions. Of the 12 candidate reference genes, SPRY domain protein (SPRYp), alpha-tubulin (TUBα), cyclophilin (CYP), L-asparaginase (L-asp), and MSF1-domain-containing protein (MSF1) were the most stably expressed under different experimental conditions, while 18S ribosomal RNA (18S), 28S ribosomal RNA (28S), and beta-actin (ACTB) were the least stably expressed. This investigation not only revealed potential factors influencing the suitability of reference genes, but also identified optimal reference genes from a pool of candidate genes under a wide range of conditions.

scholarly journals Selection of reference genes for gene expression analysis in Liriodendron hybrids’ somatic embryogenesis and germinative tissues

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Tingting Li ◽  
Weigao Yuan ◽  
Shuai Qiu ◽  
Jisen Shi
Keyword(s):  
Gene Expression ◽  
Somatic Embryogenesis ◽  
Reference Genes ◽  
Gene Selection ◽  
Elongation Factor ◽  
Reverse Transcription Pcr ◽  
Expression Of Genes ◽  
Suitable Reference ◽  
Functional Analyses ◽  
Elongation Factor 1

AbstractThe differential expression of genes is crucial for plant somatic embryogenesis (SE), and the accurate quantification of gene expression levels relies on choosing appropriate reference genes. To select the most suitable reference genes for SE studies, 10 commonly used reference genes were examined in synchronized somatic embryogenic and subsequent germinative cultures of Liriodendron hybrids by using quantitative real-time reverse transcription PCR. Four popular normalization algorithms: geNorm, NormFinder, Bestkeeper and Delta-Ct were used to select and validate the suitable reference genes. The results showed that elongation factor 1-gamma, histone H1 linker protein, glyceraldehyde-3-phosphate dehydrogenase and α-tubulin were suitable for SE tissues, while elongation factor 1-gamma and actin were best for the germinative organ tissues. Our work will benefit future studies of gene expression and functional analyses of SE in Liriodendron hybrids. It is also serves as a guide of reference gene selection in early embryonic gene expression analyses for other woody plant species.

scholarly journals Identification of the most suitable reference gene for gene expression studies with development and abiotic stress response in Bromus sterilis

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Madhab Kumar Sen ◽  
Kateřina Hamouzová ◽  
Pavlina Košnarová ◽  
Amit Roy ◽  
Josef Soukup
Keyword(s):  
Reference Gene ◽  
Herbicide Resistance ◽  
Reference Genes ◽  
Gene Selection ◽  
High Yield ◽  
Suitable Reference Gene ◽  
Grass Species ◽  
Experimental Conditions ◽  
Qrt Pcr ◽  
Suitable Reference

AbstractBromus sterilis is an annual weedy grass, causing high yield losses in winter cereals. Frequent use of herbicides had led to the evolution of herbicide resistance in this species. Mechanisms underlying herbicide resistance in B. sterilis must be uncovered because this problem is becoming a global threat. qRT-PCR and the next-generation sequencing technologies can elucidate the resistance mechanisms. Although qRT-PCR can calculate precise fold changes, its preciseness depends on the expression of reference genes. Regardless of stable expression in any given condition, no gene can act as a universal reference gene. Hence, it is necessary to identify the suitable reference gene for each species. To our knowledge, there are no reports on the suitable reference gene in any brome species so far. Thus, in this paper, the stability of eight genes was evaluated using qRT-PCR experiments followed by expression stability ranking via five most commonly used software for reference gene selection. Our findings suggest using a combination of 18S rRNA and ACCase to normalise the qRT-PCR data in B. sterilis. Besides, reference genes are also recommended for different experimental conditions. The present study outcomes will facilitate future molecular work in B. sterilis and other related grass species.

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