Validation of reference genes for gene expression studies in the emerald ash borer (<i>Agrilus planipennis</i>)

Rajarapu, Swapna Priya; Mamidala, Praveen; Mittapalli, Omprakash

doi:10.1111/j.1744-7917.2011.01447.x

Cited by 45 publications

(39 citation statements)

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“…The act gene exhibited high variation in expression among different castes and tissues with P = 0.002 and P <0.001, respectively. These results are in accordance with several studies demonstrating act gene as an unsuitable internal control for RT-PCR in other cell types or species [40], [44]. Recent studies have shown that β-act (one of the three main groups of act isoform, α, β and γ) expression can change in response to variety of conditions [45].…”

Section: Discussionsupporting

confidence: 92%

“…Based on the rankings from RefFinder, which integrates outcomes of three major statistic algorithms (geNorm, Normfinder and Bestkeeper) and assigns an appropriate weight to an individual gene and calculates the geometric mean of their weight, rpl18 and ef1-beta genes had a good performance under specific conditions. Moreover, ef1 (including ef1-alpha and ef1-beta ) had also been proved to be the best candidate reference gene for normalization in emerald ash borer [40], triatomines [41], bed bug [6] and honey bee [8].…”

Section: Discussionmentioning

confidence: 99%

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Validation of Reference Genes in Solenopsis invicta in Different Developmental Stages, Castes and Tissues

et al. 2013

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To accurately assess gene expression levels, it is essential to normalize real-time quantitative PCR (RT-qPCR) data with suitable internal reference genes. For the red imported fire ant, Solenopsis invicta, reliable reference genes to assess the transcript expression levels of the target genes have not been previously investigated. In this study, we examined the expression levels of five candidate reference genes (rpl18, ef1-beta, act, GAPDH, and tbp) in different developmental stages, castes and tissues of S. invicta. To evaluate the suitability of these genes as endogenous controls, three software-based approaches (geNorm, BestKeeper and NormFinder) and one web-based comprehensive tool (RefFinder) were used to analyze and rank the tested genes. Furthermore, the optimal number of reference gene(s) was determined by the pairwise variation value. Our data showed that two of the five candidate genes, rpl18 and ef1-beta, were the most suitable reference genes because they have the most stable expression among different developmental stages, castes and tissues in S. invicta. Although widely used as reference gene in other species, in S. invicta the act gene has high variation in expression and was consequently excluded as a reliable reference gene. The two validated reference genes, rpl18 and ef1-beta, can be widely used for quantification of target gene expression with RT-qPCR technology in S. invicta.

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Section: Discussionsupporting

confidence: 92%

Section: Discussionmentioning

confidence: 99%

Validation of Reference Genes in Solenopsis invicta in Different Developmental Stages, Castes and Tissues

et al. 2013

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“…However, in the present study, ACT was one of the least stable genes, in both non-infected and infected plants. The expression instability of ACT under specific conditions has been reported in other studies (Guénin et al, 2009;Huis et al, 2010;Rajarapu et al, 2012). Tubulin has also been shown to be unstable in some Citrus genotypes (Yan et al, 2012), during the developmental stages of flax (Huis et al, 2010), and in Orobanche ramosa (González-Verdejo et al, 2008).…”

Section: Discussionsupporting

confidence: 64%

Selection of reference genes for expression analyses of red-fleshed sweet orange (Citrus sinensis)

et al. 2015

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ABSTRACT. Red-fleshed oranges (Citrus sinensis) contain high levels of carotenoids and lycopene. The growing consumer demand for products with health benefits has increased interest in these types of Citrus cultivars as a potential source of nutraceuticals. However, little is known about the physiology of these cultivars under Brazilian conditions. Transcriptome and gene expression analyses are important tools in the breeding and management of red-fleshed sweet orange cultivars. Reverse transcription quantitative polymerase chain reaction is a method of quantifying gene expression, but various standardizations are required to obtain precise, accurate, and specific results. Among the standardizations required, the choice of suitable stable reference genes is fundamental. The objective of this study was to evaluate the stability of 11 candidate genes using various tissue and organ samples from healthy plants or leaves from citrus greening disease (Huanglongbing)-symptomatic plants of a Brazilian red-fleshed cultivar ('Sanguínea de Mombuca'), in order to select the most suitable reference gene for investigating gene expression under these conditions. geNorm and NormFinder identified genes that encoded translation initiation 18441 Selection of reference genes for red-fleshed sweet orange ©FUNPEC-RP www.funpecrp.com.br Genetics and Molecular Research 14 (4): 18440-18451 (2015) factor 3, ribosomal protein L35, and translation initiation factor 5A as the most stable genes under the biological conditions tested, and genes coding actin (ACT) and the subunit of the PSI reaction center subunit III were the least stable. Phosphatase, malate dehydrogenase, and ACT were the most stable genes in the leaf samples of infected plants.

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“…The curve consisted of 5-fold dilutions over four points. An elongation factor 1-α ( Ap EF-1 α) was used as internal control gene, which showed stable expression in A. planipennis [111]. Fold change in gene expression among male and female antennal samples of A. planipennis were derived by the 2 −δδC T method by taking newly eclosed virgin samples (male and female) as calibrator [112].…”

Section: Methodsmentioning

confidence: 99%

Identification of Odor-Processing Genes in the Emerald Ash Borer, Agrilus planipennis

Mamidala

Wijeratne

et al. 2013

PLoS ONE

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BackgroundInsects rely on olfaction to locate food, mates, and suitable oviposition sites for successful completion of their life cycle. Agrilus planipennis Fairmaire (emerald ash borer) is a serious invasive insect pest that has killed tens of millions of North American ash (Fraxinus spp) trees and threatens the very existence of the genus Fraxinus. Adult A. planipennis are attracted to host volatiles and conspecifics; however, to date no molecular knowledge exists on olfaction in A. planipennis. Hence, we undertook an antennae-specific transcriptomic study to identify the repertoire of odor processing genes involved in A. planipennis olfaction.Methodology and Principal FindingsWe acquired 139,085 Roche/454 GS FLX transcriptomic reads that were assembled into 30,615 high quality expressed sequence tags (ESTs), including 3,249 isotigs and 27,366 non-isotigs (contigs and singletons). Intriguingly, the majority of the A. planipennis antennal transcripts (59.72%) did not show similarity with sequences deposited in the non-redundant database of GenBank, potentially representing novel genes. Functional annotation and KEGG analysis revealed pathways associated with signaling and detoxification. Several odor processing genes (9 odorant binding proteins, 2 odorant receptors, 1 sensory neuron membrane protein and 134 odorant/xenobiotic degradation enzymes, including cytochrome P450s, glutathione-S-transferases; esterases, etc.) putatively involved in olfaction processes were identified. Quantitative PCR of candidate genes in male and female A. planipennis in different developmental stages revealed developmental- and sex-biased expression patterns.Conclusions and SignificanceThe antennal ESTs derived from A. planipennis constitute a rich molecular resource for the identification of genes potentially involved in the olfaction process of A. planipennis. These findings should help in understanding the processing of antennally-active compounds (e.g. 7-epi-sesquithujene) previously identified in this serious invasive pest.

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Validation of reference genes for gene expression studies in the emerald ash borer (Agrilus planipennis)

Cited by 45 publications

References 16 publications

Validation of Reference Genes in Solenopsis invicta in Different Developmental Stages, Castes and Tissues

Validation of Reference Genes in Solenopsis invicta in Different Developmental Stages, Castes and Tissues

Selection of reference genes for expression analyses of red-fleshed sweet orange (Citrus sinensis)

Identification of Odor-Processing Genes in the Emerald Ash Borer, Agrilus planipennis

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