Reference Gene Selection in<i>Phenacoccus solenopsis</i>Tinsley (Hemiptera: Pseudococcidae) and Their Normalization Impact on Gene Expression in RNAi Studies

Singh, Satnam; Pandher, Suneet; Gupta, Mona; Kaur, Gurmeet; Rathore, Pankaj

doi:10.1093/jee/toy328

Cited by 7 publications

(5 citation statements)

References 30 publications

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“…Gene functional studies were first validated through preliminary studies taking substantial number of adult mealybugs (25–30) for each gene. Thereafter the final studies were conducted with adult mealybugs in three biological replicates (5 insects/replicate) injected individually with 10 µg of gene specific dsRNA from dorsal side using glass capillary (#3-000-203-G) (Drummond Scientific, Broomall, PA) and Nanojet TM (Drummond Scientific) 22 . The same quantity of dsGFP was injected in control insects.…”

Section: Methodsmentioning

confidence: 99%

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Using de novo transcriptome assembly and analysis to study RNAi in Phenacoccus solenopsis Tinsley (Hemiptera: Pseudococcidae)

Singh

Gupta

Pandher

et al. 2019

Sci Rep

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Phenacoccus solenopsis is one of the major polyphagous crop pests in India. Inadequate genomic or transcriptomic resources have limited the molecular studies in this insect despite its huge economic importance. The existing molecular sequence resources of this insect were supplemented through RNA sequencing, de novo transcriptome assembly and analysis, which generated 12, 925 CDS from 23,643 contigs with an average size of 1077.5 bp per CDS and 85.1% positive BLAST hits with NCBI Non redundant (nr) database. Twenty three genes involved in RNAi machinery identified through BLASTx search against NCBI nr database suggested the existence of robust RNAi in mealybug. RNAi in P. solenopsis was demonstrated through knockdown of IAP (Inhibitor of Apoptosis), AQP (Aquaporin), CAL (Calcitonin), VATPase (V-type proton ATPase subunit F 1), bursicon, chitin synthase, SNF7 and α-amylase by injecting sequence specific dsRNA of respective genes in adult female. Additionally, feeding RNAi has been demonstrated in 2nd instar nymph through dsRNA uptake in plant. The knockdown of core RNAi machinery genes such as Dicer, Argonaute and Staufen significantly hampered RNAi efficiency in this insect. However, downregulation of dsRNases improved RNAi efficiency. Sequential studies for understanding RNAi in P. solenopsis using transcriptome sequences have also been reported. The present study provides a base for future research on developing RNAi as strategy for management of this pest.

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Section: Methodsmentioning

confidence: 99%

“…Each qPCR reaction constituted of 1:10 diluted 1 µl of cDNA, 0.2 µl of gene-specific primers (10 mM) (Table 1) and 5 µl of SYBR premix and rest nuclease free water to make final volume of 10 µl. The relative expression level of each gene was estimated using ∆∆CT method after normalization with 28 s (MH712871.1) as reference gene 22 .…”

Section: Methodsmentioning

confidence: 99%

Using de novo transcriptome assembly and analysis to study RNAi in Phenacoccus solenopsis Tinsley (Hemiptera: Pseudococcidae)

Singh

Gupta

Pandher

et al. 2019

Sci Rep

Self Cite

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“…0 expression was inconsistent when normalized to the least stable reference compared with that when normalized to the optimal reference set or the optimal reference alone. These findings revealed that the arbitrary selection of reference genes would lead to inaccurate or contradictory results for target genes [ 15 , 41 ], and our results demonstrated that the combined use of optimal reference genes ensures greater accuracy in gene expression analysis.…”

Section: Discussionmentioning

confidence: 86%

Evaluation of candidate reference genes for gene expression analysis in the brassica leaf beetle, Phaedon brassicae (Coleoptera: Chrysomelidae)

Jiang

Liu

et al. 2021

PLoS ONE

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The brassica leaf beetle Phaedon brassicae is a notorious defoliator of cruciferous vegetables. However, few molecular studies of this pest have been conducted due to limited sequence data. Recently, RNA sequencing has offered a powerful platform to generate numerous transcriptomic data, which require RT-qPCR to validate target gene expression. The selection of reliable reference genes to normalize RT-qPCR data is a prerequisite for gene expression analysis. In the present study, the expression stabilities of eight candidate reference genes under biotic conditions (development stages and various tissues) and abiotic perturbations (thermal stress and pesticide exposure) were evaluated using four different statistical algorithms. The optimal suites of reference genes were recommended for the respective experimental conditions. For tissue expression analysis, RPL32 and EF-1α were recommended as the suitable reference genes. RPL19 and TBP were the optimal reference genes across different developmental stages. RPL32 and TBP were identified as the most suitable references for thermal stress. Furthermore, RPL32 and RPL19 were ranked as the best references for insecticide exposure. This work provides a systematic exploration of the optimal reference genes for the respective experimental conditions, and our findings would facilitate molecular studies of P. brassicae.

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“…The qRT-PCR is a reliable technique in gene expression analysis, with high sensitivity and specificity, and the qRT-PCR data must be normalized by suitable reference genes to avoid expression differences among samples [31]. Recently, there have many reports on reference genes selection of various insects under different abiotic and biotic conditions [32][33][34][35][36], including S. frugiperda [27]. In these studies, the expression levels of conventional reference genes in different insects are quite different, and none of them with similar expression levels under all conditions, which indicates that there is no absolute universality among homologous reference genes.…”

Section: Discussionmentioning

confidence: 99%

Selection and Evaluation of Reference Genes for qRT-PCR in Spodoptera frugiperda (Lepidoptera: Noctuidae)

Han

Qin

et al. 2021

Insects

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As an accurate and convenient technique, the qRT-PCR is always used in the quantitative expression analysis of functional genes. Normalization of the data relies on stable reference genes. The fall armyworm Spodoptera frugiperda (J. E. Smith) is an important invasive and migratory pest that seriously threatens corn production around the world. In this paper, we selected 10 candidate reference genes (18S, AK, RPL10, RPS24, 28S, SOD, ATP, GAPDH, ACT, and a-TUB) and determined their expression levels under different conditions (different developmental stages, various tissues, mating status, hormones, diets, and temperatures). Subsequently, the stability of reference genes was evaluated by four algorithms (Delta Ct method, geNorm, NormFinder, BestKeeper). The optimal combination of reference genes for each treatment was obtained by geNorm. Finally, the comprehensive ranks were determined by the online tool RefFinder. Results showed that the most stable reference genes were SOD, RPL10, and RPS24 for developmental stages, α-TUB, RPL10, and ATP for different tissues, AK, RPL10, and 18S for mating status, 18S and AK under hormone treatment, 18S, RPL10, and SOD under diet treatment, RPL10, 18S, and RPS24 under temperature treatment. This study confirmed recent data on a few reference genes and provided an evaluation of a number of additional reference genes of S. frugiperda under various conditions.

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Reference Gene Selection inPhenacoccus solenopsisTinsley (Hemiptera: Pseudococcidae) and Their Normalization Impact on Gene Expression in RNAi Studies

Cited by 7 publications

References 30 publications

Using de novo transcriptome assembly and analysis to study RNAi in Phenacoccus solenopsis Tinsley (Hemiptera: Pseudococcidae)

Using de novo transcriptome assembly and analysis to study RNAi in Phenacoccus solenopsis Tinsley (Hemiptera: Pseudococcidae)

Evaluation of candidate reference genes for gene expression analysis in the brassica leaf beetle, Phaedon brassicae (Coleoptera: Chrysomelidae)

Selection and Evaluation of Reference Genes for qRT-PCR in Spodoptera frugiperda (Lepidoptera: Noctuidae)

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