Dataset Information

Identification and Validation of Reference Genes for the Normalization of Gene Expression Data in qRT-PCR Analysis in Aphis gossypii (Hemiptera: Aphididae).

ABSTRACT: To obtain accurate and reliable results from quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) analysis, it is necessary to select suitable reference genes as standards for normalizing target gene expression data. QRT-PCR is a popular analytical methodology for studying gene expression and it has been used widely in studies of Aphis gossypii Glover in recent years. However, there is absence of study on the stability of the expression of reference genes in A. gossypii. In this study, eight commonly used candidate reference genes, including 18S, 28S, ?-ACT, GAPDH, EF1?, RPL7, ?-TUB, and TBP, were evaluated under various experimental conditions to assess their suitability for use in the normalization of qRT-PCR data. The optimal number of reference genes was determined using the geNorm program, and the suitability of particular reference genes was empirically validated by performing normalizations of expression data for the HSP70 gene. The results showed the most suitable combinations of reference genes for the different experimental conditions. For experiments based on divergent developmental stages, EF1?, ?-ACT, and RPL7 are the optimal reference gene combination, both EF1? and ?-ACT are the optimal combination used in the experiments of different geographical populations, whereas for experiments of the temperature changes, the combination of GAPDH and RPL7 is optimal, both 18S and ?-ACT are an optimal combination for feeding assay experiments. These research results should be useful for the selection of the suitable reference genes to obtain reliable qRT-PCR data in the gene expression study of A. gossypii.

SUBMITTER: Ma KS

PROVIDER: S-EPMC5778981 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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Publications

Identification and Validation of Reference Genes for the Normalization of Gene Expression Data in qRT-PCR Analysis in Aphis gossypii (Hemiptera: Aphididae).

Ma Kang-Sheng KS Li Fen F Liang Ping-Zhuo PZ Chen Xue-Wei XW Liu Ying Y Gao Xi-Wu XW

Journal of insect science (Online) 20160205 1

To obtain accurate and reliable results from quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) analysis, it is necessary to select suitable reference genes as standards for normalizing target gene expression data. QRT-PCR is a popular analytical methodology for studying gene expression and it has been used widely in studies of Aphis gossypii Glover in recent years. However, there is absence of study on the stability of the expression of reference genes in A. gossyp ...[more]

PMID: 28076279

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Project description:BackgroundDendrocalamus latiflorus Munro distributes widely in subtropical areas and plays vital roles as valuable natural resources. The transcriptome sequencing for D. latiflorus Munro has been performed and numerous genes especially those predicted to be unique to D. latiflorus Munro were revealed. qRT-PCR has become a feasible approach to uncover gene expression profiling, and the accuracy and reliability of the results obtained depends upon the proper selection of stable reference genes for accurate normalization. Therefore, a set of suitable internal controls should be validated for D. latiflorus Munro.ResultsIn this report, twelve candidate reference genes were selected and the assessment of gene expression stability was performed in ten tissue samples and four leaf samples from seedlings and anther-regenerated plants of different ploidy. The PCR amplification efficiency was estimated, and the candidate genes were ranked according to their expression stability using three software packages: geNorm, NormFinder and Bestkeeper. GAPDH and EF1α were characterized to be the most stable genes among different tissues or in all the sample pools, while CYP showed low expression stability. RPL3 had the optimal performance among four leaf samples. The application of verified reference genes was illustrated by analyzing ferritin and laccase expression profiles among different experimental sets. The analysis revealed the biological variation in ferritin and laccase transcript expression among the tissues studied and the individual plants.ConclusionsgeNorm, NormFinder, and BestKeeper analyses recommended different suitable reference gene(s) for normalization according to the experimental sets. GAPDH and EF1α had the highest expression stability across different tissues and RPL3 for the other sample set. This study emphasizes the importance of validating superior reference genes for qRT-PCR analysis to accurately normalize gene expression of D. latiflorus Munro.

Project description:Insects harbor a wide variety of microorganisms that form complex and changing communities and play an important role in the biology and evolution of their hosts. Aphids have been used as model organisms to study microorganism-insect interactions. Almost all aphids are infected with the obligate endosymbiont Buchnera aphidicola and can host different bacteria that allow them to acquire traits of agronomic importance, such as resistance to high temperatures and/or defense against natural enemies. However, the bacterial communities of most aphid species remain poorly characterized. In this study, we used high-throughput DNA sequencing to characterize the bacterial communities of Aphis gossypii and Myzus persicae from two cultivable pepper species, Capsicum frutescens (Tabasco variety) and C. annuum (Cayenne variety), in four localities of southwestern Colombia. In addition, we evaluated the dynamics of A. gossypii-associated microorganisms on a seasonal basis. Our results show that the bacterial communities of A. gossypii and M. persicae are dominated by the primary endosymbiont B. aphidicola, while the presence of the facultative symbiont Arsenophonus sp. was only detected in one A. gossypii population from cayenne pepper. In addition to these two known symbionts, eight bacterial OTUs were identified that presented a frequency of 1% or more in at least one of the analyzed populations. The results show that the bacterial communities of aphids associated with pepper crops appears to be structured according to the host aphid species and the geographical location, while no differences were observed in the diversity of bacteria between host plants. Finally, the diversity and abundance of the A. gossypii bacterial community was variable among the four sampling points evaluated over the year and showed a relation with the aphid's population dynamics. This study represents the first approach to the knowledge of the bacterial community present in chili pepper aphids from Colombia. Nevertheless, more in-depth studies, including replicates, are required to confirm the patterns observed in the microbial communities of aphids from pepper crops.

Dataset Information

Identification and Validation of Reference Genes for the Normalization of Gene Expression Data in qRT-PCR Analysis in Aphis gossypii (Hemiptera: Aphididae).

Publications

Identification and Validation of Reference Genes for the Normalization of Gene Expression Data in qRT-PCR Analysis in Aphis gossypii (Hemiptera: Aphididae).

Similar Datasets

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