Evaluation of Appropriate Reference Genes For Investigating Gene Expression in Chlorops oryzae (Diptera: Chloropidae)

2019 ◽  
Vol 112 (5) ◽  
pp. 2207-2214 ◽  
Author(s):  
Ping Tian ◽  
Lin Qiu ◽  
Ailin Zhou ◽  
Guo Chen ◽  
Hualiang He ◽  
...  
Keyword(s):  
Gene Expression ◽  
Ribosomal Protein ◽  
Reference Genes ◽  
Molecular Mechanisms ◽  
Developmental Stages ◽  
Quantitative Polymerase Chain Reaction ◽  
Housekeeping Genes ◽  
Future Research ◽  
Economic Damage ◽  
Physiological Processes

Abstract Reverse transcription quantitative polymerase chain reaction (PCR) has become an invaluable technique for analyzing gene expression in many insects. However, this approach requires the use of stable reference genes to normalize the data. Chlorops oryzae causes significant economic damage to rice crops throughout Asia. The lack of suitable reference genes has hindered research on the molecular mechanisms underlying many physiological processes of this species. In this study, we used quantitative real-time PCR to evaluate the expression of eight C. oryzae housekeeping genes glyceraldehyde-3-phosphate dehydrogenase (GAPDH), β-actin (βACT), beta-tubulin (βTUB), Delta Elongation factor-1 (EF1δ), ribosomal protein S11 (RPS11), RPS15, C-terminal-Binding Protein (CtBP), and ribosomal protein 49 (RP49) in different developmental stages and tissues in both larvae and adults. We analyzed the data with four different software packages: geNorm, NormFinder, BestKeeper, and RefFinder and compared the results obtained with each method. The results indicate that PRS15 and RP49 can be used as stable reference genes for quantifying gene expression in different developmental stages and larval tissues. GAPDH and βACT, which have been considered stable reference genes by previous studies, were the least stable of the candidate genes with respect to larval tissues. GAPDH was, however, the most stable reference gene for adult tissues. We verified the candidate reference genes identified and found that the expression levels of Cadherins (Cads) changed when different reference genes were used to normalize gene expression. This study provides a valuable foundation for future research on gene function, and investigating the molecular basis of physiological processes, in C. oryzae.

scholarly journals Selection and Validation of Reference Genes for Gene Expression Analysis in Tuta absoluta Meyrick (Lepidoptera: Gelechiidae)

Insects ◽  
2021 ◽  
Vol 12 (7) ◽  
pp. 589
Author(s):  
Xin Yan ◽  
Yibo Zhang ◽  
Kangkang Xu ◽  
Yawei Wang ◽  
Wenjia Yang
Keyword(s):  
Gene Expression ◽  
Reference Genes ◽  
Developmental Stages ◽  
Quantitative Polymerase Chain Reaction ◽  
Housekeeping Genes ◽  
Tuta Absoluta ◽  
Leaf Miner ◽  
Specific Reference ◽  
Internal Reference ◽  
Experimental Conditions

The tomato leaf miner, Tuta absoluta is a destructive pest of tomato. The leaf-mining activities of its larvae can cause significant yield losses. Real-time quantitative polymerase chain reaction (RT-qPCR) is commonly used to measure gene expression, and the selection of stable reference genes for calibration and standardization is critical for accurate use of RT-qPCR. We studied the stable expression of nine common housekeeping genes in T. absoluta. These were examined at different developmental stages, in larval tissues, as well as those induced by exposure to 20E and insecticides. Four dedicated algorithms (geNorm, BestKeeper, NormFinder, and ΔCt method) and online tool (RefFinder) were used to analyze and rank the tested reference genes. Based on the standardized gene expression data of target gene ecdysone receptor (EcR), the applicability of specific reference genes was verified. The results clarify that the optimal internal reference genes vary greatly under different experimental conditions. GAPDH and RPS11 were the best reference genes for developmental stages; RPL28 and RPL10 for different tissues; EF1α and RPL28 for 20E treatment; EF1α and RPL7A for insecticide treatments. The most suitable reference genes in all experimental conditions are EF1α and RPL28.

scholarly journals Identification of appropriate reference genes for gene expression studies in mice left ventricles from different developmental stages

2021 ◽  
Author(s):  
Jie Ren ◽  
Ningning Zhang ◽  
Xiangjie Li ◽  
Xiaogang Sun ◽  
Jiangping Song
Keyword(s):  
Gene Expression ◽  
Heart Development ◽  
Reference Genes ◽  
Developmental Stages ◽  
Quantitative Polymerase Chain Reaction ◽  
Expression Patterns ◽  
Housekeeping Genes ◽  
Good Choice ◽  
Expression Stability ◽  
Gene Expressions

Abstract Aims: Real-time quantitative polymerase chain reaction (RT-qPCR) is the standard assay used for revealing the gene expression characteristics. However, the RT-qPCR studies all need reference genes for normalization to make the results comparable, which should hold a high expression stability during all experimental datasets. So far, there was no optimal set of reference genes identified in mice left ventricles (LV) across embryonic and postnatal stages. The objective of our research was to identify the appropriate reference genes in mice LV from different developmental stages.Methods and Results: we investigated the gene expressions of common 21 candidate housekeeping genes in mice LV from 7 different developmental stages, almost throughout the whole period of the mouse lifespan. The expression of some candidate reference genes, such as 18S and Actb, apparently fluctuated. The stability of potential reference genes was evaluated by a number of methods, such as GeNorm, NormFinder, BestKeeper, Delta-Ct and RefFinder method. we identified a set of optimal reference genes that can be reliably used for normalization of RT-qPCR experiments in different developmental stages of mice LV. Our results showed that following genes should not be used as reference genes in mice LV development studies: 18S, Hmbs, Ubc, Psmb4, Tfrc and Actb. And the Rplp0 appeared to represent a good choice. Conclusions: Our study provides the expression stability of the commonly used reference genes in process of LV development and maturation. We also identified a set of optimal reference genes under different conditions. Our findings may be helpful in future studies to investigate the gene expression patterns and mechanism of mammalian heart development.

scholarly journals Comparative Transcriptomic Analysis of Riptortus pedestris (Hemiptera: Alydidae) to Characterize Wing Formation across All Developmental Stages

Insects ◽  
2021 ◽  
Vol 12 (3) ◽  
pp. 226
Author(s):  
Siying Fu ◽  
Yujie Duan ◽  
Siqi Wang ◽  
Yipeng Ren ◽  
Wenjun Bu
Keyword(s):  
Molecular Mechanisms ◽  
Developmental Stages ◽  
Average Length ◽  
Transcriptome Assembly ◽  
Expression Profiles ◽  
Economic Losses ◽  
Future Research ◽  
Economic Damage ◽  
Wing Formation ◽  
Riptortus Pedestris

Riptortus pedestris (Hemiptera: Alydidae) is a major agricultural pest in East Asia that causes considerable economic losses to the soybean crop each year. However, the molecular mechanisms governing the growth and development of R. pedestris have not been fully elucidated. In this study, the Illumina HiSeq6000 platform was employed to perform de novo transcriptome assembly and determine the gene expression profiles of this species across all developmental stages, including eggs, first-, second-, third-, fourth-, and fifth-instar nymphs, and adults. In this study, a total of 60,058 unigenes were assembled from numerous raw reads, exhibiting an N50 length of 2126 bp and an average length of 1199 bp, and the unigenes were annotated and classified with various databases, such as the Kyoto Encyclopedia of Genes and Genomes (KEGG), Clusters of Orthologous Groups (COG), and Gene Ontology (GO). Furthermore, various numbers of differentially expressed genes (DEGs) were calculated through pairwise comparisons of all life stages, and some of these DEGs were associated with immunity, metabolism, and development by GO and KEGG enrichment. In addition, 35,158 simple sequence repeats (SSRs) and 715,604 potential single nucleotide polymorphisms (SNPs) were identified from the seven transcriptome libraries of R. pedestris. Finally, we identified and summarized ten wing formation-related signaling pathways, and the molecular properties and expression levels of five wing development-related genes were analyzed using quantitative real-time PCR for all developmental stages of R. pedestris. Taken together, the results of this study may establish a foundation for future research investigating developmental processes and wing formation in hemimetabolous insects and may provide valuable data for pest control efforts attempting to reduce the economic damage caused by this pest.

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.

scholarly journals Reference genes for RT-qPCR normalisation in different tissues, developmental stages and stress conditions of Hypericum perforatum

2018 ◽  
Author(s):  
Zhezhi Wang ◽  
Wen Zhou ◽  
Lei Yang ◽  
Yan Sun ◽  
Qian Zhang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Candidate Genes ◽  
Reference Genes ◽  
Hypericum Perforatum ◽  
Developmental Stages ◽  
Housekeeping Genes ◽  
Potential Candidate ◽  
Qrt Pcr ◽  
Wide Range ◽  
Different Tissues

Hypericum perforatum is a widely known medicinal herb used mostly as a remedy for depression because of its abundant secondary metabolites. Quantitative real-time PCR (qRT-PCR) is an optimized method for the efficient and reliable quantification of gene expression studies. In general, reference genes are used in qRT-PCR analysis because of their known or suspected housekeeping roles. However, their expression level cannot be assumed to remain stable under all possible experimental conditions. Thus, the identification of high quality reference genes is very necessary for the interpretation of qRT-PCR data. In this study, we investigated the expression of fourteen candidate genes, including nine housekeeping genes and five potential candidate genes. Additionally, the HpHYP1 gene, belonging to the PR-10 family associated with stress control, was used for validation of the candidate reference genes. Three programs were applied to evaluate the gene expression stability across four different plant tissues, three developmental stages and a set of abiotic stress and hormonal treatments. The candidate genes showed a wide range of Ct values in all samples, indicating that they are differentially expressed. Integrating all of the algorithms and evaluations, ACT2 and TUB-β were the most stable combination overall and for different developmental stages samples. Moreover, ACT2 and EF1-α were considered to be the two most applicable reference genes for different tissues and for stress samples. Majority of the conventional housekeeping genes exhibited better than the potential reference genes. The obtained results will contribute to improving credibility of standardization and quantification of transcription levels in future expression research of H. perforatum.

Screening of reference genes using real-time quantitative PCR for gene expression studies inNeoseiulus barkeriHughes (Acari: Phytoseiidae)

2018 ◽  
Vol 109 (4) ◽  
pp. 443-452 ◽  
Author(s):  
C. Wang ◽  
J. Yang ◽  
Q. Pan ◽  
S. Yu ◽  
R. Luo ◽  
...  
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Reference Genes ◽  
Developmental Stages ◽  
Quantitative Polymerase Chain Reaction ◽  
Real Time Quantitative Pcr ◽  
Expression Studies ◽  
Gene Expression Studies ◽  
The Stability ◽  
Suitable Reference

AbstractA stable reference gene is a key prerequisite for accurate assessment of gene expression. At present, the real-time reverse transcriptase quantitative polymerase chain reaction has been widely used in the analysis of gene expression in a variety of organisms.Neoseiulus barkeriHughes (Acari: Phytoseiidae) is a major predator of mites on many important economically crops. Until now, however, there are no reports evaluating the stability of reference genes in this species. In view of this, we used GeNorm, NormFinder, BestKeeper, and RefFinder software tools to evaluate the expression stability of 11 candidate reference genes in developmental stages and under various abiotic stresses. According to our results, β-ACTandHsp40were the top two stable reference genes in developmental stages. TheHsp60andHsp90were the most stable reference genes in various acaricides stress. For alterations in temperature,Hsp40and α-TUBwere the most suitable reference genes. About UV stress,EF1α and α-TUBwere the best choice, and for the different prey stress, β-ACTand α-TUBwere best suited. In normal conditions, the β-ACT and α-TUB were the two of the highest stable reference genes to respond to all kinds of stresses. The current study provided a valuable foundation for the further analysis of gene expression inN. barkeri.

scholarly journals Evaluation of potential reference genes for real-time qPCR analysis in a biparental beetle,Lethrus apterus(Coleoptera: Geotrupidae)

PeerJ ◽  
2017 ◽  
Vol 5 ◽  
pp. e4047 ◽  
Author(s):  
Nikoletta A. Nagy ◽  
Zoltán Németh ◽  
Edit Juhász ◽  
Szilárd Póliska ◽  
Rita Rácz ◽  
...  
Keyword(s):  
Ribosomal Protein ◽  
Real Time ◽  
Reference Genes ◽  
Hormonal Regulation ◽  
Quantitative Polymerase Chain Reaction ◽  
Expression Patterns ◽  
Housekeeping Genes ◽  
Body Region ◽  
Body Regions ◽  
Polymerase Chain

Hormones play an important role in the regulation of physiological, developmental and behavioural processes. Many of these mechanisms in insects, however, are still not well understood. One way to investigate hormonal regulation is to analyse gene expression patterns of hormones and their receptors by real-time quantitative polymerase chain reaction (RT-qPCR). This method, however, requires stably expressed reference genes for normalisation. In the present study, we evaluated 11 candidate housekeeping genes as reference genes in samples ofLethrus apterus, an earth-boring beetle with biparental care, collected from a natural population. For identifying the most stable genes we used the following computational methods: geNorm, NormFinder, BestKeeper, comparative delta Ct method and RefFinder. Based on our results, the two body regions sampled (head and thorax) differ in which genes are most stably expressed. We identified two candidate reference genes for each region investigated: ribosomal protein L7A and RP18 in samples extracted from the head, and ribosomal protein L7A and RP4 extracted from the muscles of the thorax. Additionally, L7A and RP18 appear to be the best reference genes for normalisation in all samples irrespective of body region. These reference genes can be used to study the hormonal regulation of reproduction and parental care inLethrus apterusin the future.

scholarly journals Identification of reference genes for gene expression studies among different developmental stages of murine hearts

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Jie Ren ◽  
Ningning Zhang ◽  
Xiangjie Li ◽  
Xiaogang Sun ◽  
Jiangping Song
Keyword(s):  
Gene Expression ◽  
Reference Gene ◽  
Heart Development ◽  
Reference Genes ◽  
Developmental Stages ◽  
Expression Profiles ◽  
Quantitative Polymerase Chain Reaction ◽  
Expression Patterns ◽  
Gene Expression Profiles ◽  
Qpcr Data

Abstract Background Real-time quantitative polymerase chain reaction (RT-qPCR) is a widely-used standard assay for assessing gene expression. RT-qPCR data requires reference genes for normalization to make the results comparable. Therefore, the selected reference gene should be highly stable in its expression throughout the experimental datasets. So far, reports about the optimal set of reference genes in murine left ventricle (LV) across embryonic and postnatal stages are few. The objective of our research was to identify the appropriate reference genes in murine LV among different developmental stages. Methods We investigated the gene expression profiles of 21 widely used housekeeping genes in murine LV from 7 different developmental stages (almost throughout the whole period of the mouse lifespan). The stabilities of the potential reference genes were evaluated by five methods: GeNorm, NormFinder, BestKeeper, Delta-Ct and RefFinder. Results We proposed a set of reliable reference genes for normalization of RT-qPCR experimental data in different conditions. Furthermore, our results showed that 6 genes (18S, Hmbs, Ubc, Psmb4, Tfrc and Actb) are not recommended to be used as reference genes in murine LV development studies. The data also suggested that the Rplp0 gene might serve as an optimal reference gene in gene expression analysis. Conclusions Our study investigated the expression stability of the commonly used reference genes in process of LV development and maturation. We proposed a set of optimal reference genes that are suitable for accurate normalization of RT-qPCR data in specific conditions. Our findings may be helpful in future studies for investigating the gene expression patterns and mechanism of mammalian heart development.

scholarly journals A Scientific Note of Housekeeping Genes for the Primitively Eusocial bee Euglossa viridissima Friese (Apidae: Euglossini)

Sociobiology ◽  
2018 ◽  
Vol 65 (4) ◽  
pp. 766
Author(s):  
Samuel Boff ◽  
Anna Friedel ◽  
Anja Miertsch ◽  
J. Javier Quezada-Euàn ◽  
Robert J Paxton ◽  
...  
Keyword(s):  
Gene Expression ◽  
Expression Pattern ◽  
Reference Genes ◽  
Developmental Stages ◽  
Gene Expression Pattern ◽  
Housekeeping Genes ◽  
Expression Of Genes ◽  
Gene Expression Studies ◽  
The Stability ◽  
Primitively Eusocial

Studies on the expression of genes in different contexts are essential to our understanding of the functioning of organisms and their adaptations to the environment. Gene expression studies require steps of normalization, which are done using the stable expression pattern of reference genes. For many different eusocial bees reference genes have been discovered, but not for the primitively eusocial euglossine bees.We used available genomic resources of euglossine species and the gene information of Apis melliferato develop a set of reference genes for the primitive eusocial bee Euglossaviridissima. We tested nine genes in distinct developmental stages three different algorithms to infer the stability of gene expression. The Tata binding protein(Tbp) and 14-3-3epsilon were the most stable genes across all different stages. The strongest deviation in gene expression pattern occurred in pupae, which require a different set of genes for normalizing gene expression. 

scholarly journals Evaluation of potential reference genes for real-time qPCR analysis in a biparental beetle, Lethrus apterus (Coleoptera: Geotrupidae)

2017 ◽  
Author(s):  
Nikoletta A Nagy ◽  
Zoltán Németh ◽  
Edit Juhász ◽  
Szilárd Póliska ◽  
Rita Rácz ◽  
...  
Keyword(s):  
Ribosomal Protein ◽  
Real Time ◽  
Reference Genes ◽  
Hormonal Regulation ◽  
Quantitative Polymerase Chain Reaction ◽  
Expression Patterns ◽  
Housekeeping Genes ◽  
Body Regions ◽  
Qpcr Analysis ◽  
Polymerase Chain

Hormones play an important role in the regulation of physiological, developmental and behavioural processes. Many of these mechanisms in insects, however, are still not well understood. One way to investigate hormonal regulation is to analyse gene expression patterns of hormones and their receptors in question by real-time quantitative polymerase chain reaction (RT-qPCR). This method, however, requires stably expressed reference genes for normalisation. In the present study, we evaluated 11 candidate housekeeping genes as reference genes in samples of Lethrus apterus, an earth-boring beetle with biparental care, collected from a natural population. For identifying the most stable genes we used the following computational methods: geNorm, NormFinder, BestKeeper, comparative delta Ct method and RefFinder. Based on our results, the two body regions sampled (head and thorax) differ in which genes are most stably expressed. We identified two candidate reference genes for each region investigated: ribosomal protein L7A and RP18 in samples extracted from the head, and ribosomal protein L7A and RP4 extracted from the muscles of the thorax. These reference genes can be used to study the hormonal regulation of reproduction and parental care in Lethrus apterus in the future.

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