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.

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.

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.

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 RT-qPCR Analysis in Spinacia oleracea under Abiotic Stress

2021 ◽  
Vol 2021 ◽  
pp. 1-12
Author(s):  
Hao Xie ◽  
Bo Li ◽  
Yu Chang ◽  
Xiaoyan Hou ◽  
Yue Zhang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Expression Pattern ◽  
Reference Genes ◽  
18S Rrna ◽  
Spinacia Oleracea ◽  
Expression Patterns ◽  
Gene Expression Pattern ◽  
Housekeeping Genes ◽  
Qpcr Analysis ◽  
The Stability

Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is an accurate and convenient method for mRNA quantification. Selection of optimal reference gene(s) is an important step in RT-qPCR experiments. However, the stability of housekeeping genes in spinach (Spinacia oleracea) under various abiotic stresses is unclear. Evaluating the stability of candidate genes and determining the optimal gene(s) for normalization of gene expression in spinach are necessary to investigate the gene expression patterns during development and stress response. In this study, ten housekeeping genes, 18S ribosomal RNA (18S rRNA), actin, ADP ribosylation factor (ARF), cytochrome c oxidase subunit 5C (COX), cyclophilin (CYP), elongation factor 1-alpha (EF1α), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), histone H3 (H3), 50S ribosomal protein L2 (RPL2), and tubulin alpha chain (TUBα) from spinach, were selected as candidates in roots, stems, leaves, flowers, and seedlings in response to high temperature, CdCl2, NaCl, NaHCO3, and Na2CO3 stresses. The expression of these genes was quantified by RT-qPCR and evaluated by NormFinder, BestKeeper, and geNorm. 18S rRNA, actin, ARF, COX, CYP, EF1α, GAPDH, H3, and RPL2 were detected as optimal reference genes for gene expression analysis of different organs and stress responses. The results were further confirmed by the expression pattern normalized with different reference genes of two heat-responsive genes. Here, we optimized the detection method of the gene expression pattern in spinach. Our results provide the optimal candidate reference genes which were crucial for RT-qPCR analysis.

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