Identification and validation of potential reference gene for effective dsRNA knockdown analysis in Chilo partellus

Adeyinka, Olawale Samuel; Tabassum, Bushra; Nasir, Idrees Ahmad; Yousaf, Iqra; Sajid, Imtiaz Ahmad; Shehzad, Khurram; Batcho, Anicet; Husnain, Tayyab

doi:10.1038/s41598-019-49810-w

Cited by 16 publications

(17 citation statements)

References 48 publications

(50 reference statements)

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“…This is mainly related to the variability of reference genes in different environments and species. In addition, in the studies of reference gene stability, these commonly used reference genes are often not the most optimal choice (Chapman and Waldenström, 2015;Adeyinka et al, 2019;Gao et al, 2020), and this study has the same findings. In the qRT-PCR research on ginger, common reference genes (TUB, actin, EF1α, etc.)…”

Section: Discussionmentioning

confidence: 51%

Identification of Ginger (Zingiber officinale Roscoe) Reference Genes for Gene Expression Analysis

Liu

et al. 2020

Front. Genet.

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Quantitative real-time PCR (qRT-PCR) is widely used in the detection of gene expression level. However, there is no suitable ginger reference gene for qPCR analysis. Therefore, it is the primary task to select and validate the appropriate ginger reference gene to normalize the expression of target genes. In this study, 14 candidate reference genes were selected and analyzed in different tissues (leaf, and rhizome), different development stages, different varieties, and abiotic stress (ABA and salt stress). Expression stability was calculated using geNorm and NormFinder, Bestkeeper, and RefFinder. For abiotic stress and total conditions, 28S and COX were identified as the most stable genes. In addition, RPII was the most stable in the different development stages and different varieties. TEF2 and RPL2 were the least stably expressed in the tissue and all the conditions. In order to verify the feasibility of these genes as reference genes, we used the most stable and least stable reference genes to normalize the expression levels of ZoSPS genes under different conditions. This work can provide theoretical support for future research on ginger gene expression.

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Section: Discussionmentioning

confidence: 51%

Identification of Ginger (Zingiber officinale Roscoe) Reference Genes for Gene Expression Analysis

Liu

et al. 2020

Front. Genet.

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“…RT-qPCR is an important, simple and practical technique for assessing gene expression when compared to other quantitative methods such as Northern blotting, in situ hybridization and RNA-seq technology (Peng et al, 2018;Adeyinka et al, 2019;He et al, 2019). The application of accurate reference genes is crucial for the quantification of gene expression in A. mellifera and A. cerana.…”

Section: Discussionmentioning

confidence: 99%

Screening and Validation of Reference Genes for RT-qPCR Under Different Honey Bee Viral Infections and dsRNA Treatment

Deng¹,

Zhao²,

Yang³

et al. 2020

Front. Microbiol.

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Honey bee viruses are one of the most important pathogens that have contributed to the decrease in honey bee colony health. To analyze the infection dynamics of honey bee viruses, quantification of viral gene expression by RT-qPCR is necessary. However, suitable reference genes have not been reported from viral and RNAi studies of honey bee. Here, we evaluated the expression of 11 common reference genes (ache2, rps18, β-actin, tbp, tif, rpl32, gadph, ubc, α-tubulin, rpl14, and rpsa) from Apis mellifera (Am) and Apis cerana (Ac) under Israeli acute paralysis virus (IAPV), chronic bee paralysis virus (CBPV), and Chinese sacbrood virus (CSBV) infection as well as dsRNA-PGRP-SA treatment, and we confirmed their validation by evaluating the levels of the defensin 1 and prophenoloxidase (ppo) genes during viral infection. Our results showed that the expression of selected genes varied under different viral infections. ache2, rps18, β-actin, tbp, and tif can be used to normalize expression levels in Apis mellifera under IAPV infection, while the combination of actin and tif is suitable for CBPV-infected experiments. The combination of rpl14, tif, rpsa, ubc, and ache2 as well as more reference genes is suitable for CSBV treatment in Apis cerana. Rpl14, tif, rps18, ubc, and α-tubulin were the most stable reference genes under dsRNA treatment in Apis mellifera. Furthermore, the geNorm and NormFinder algorithms showed that tif was the best suitable reference gene for these four treatments. This study screened and validated suitable reference genes for the quantification of viral levels in honey bee, as well as for RNAi experiments.

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“…The cultures were diluted to 500 mL and incubated until it reaches OD 595 = 0.4. Then, the dsRNA synthesis was induced by the addition of 0.6 mmol l−1 IPTG at 37ºC for another 3-4 h. Raw bacteriallyexpressed dsRNAs were harvested and some of the harvested cells were purified as we described in our initial publication (Adeyinka et al 2019).…”

Section: Construction Of Dsrna Expression Vector and Inductionmentioning

confidence: 99%

A Protective dsRNA is Crucial for Optimum RNAi Gene Silencing in Chilo partellus

Adeyinka¹

2021

IJAB

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RNAi technology is currently employed as an alternate control measure for agricultural pests. However, the variability of RNAi efficiency in insect pests limits the extensive usage of this technology and demands identifying the best target gene for effective RNAi. Four different bacterially-expressed dsRNA and purified dsRNAs coated on artificial diet were fed to the larvae. The transcripts expression was analyzed at 5 days and 15 days post-exposure to various dsRNAs. In the larvae fed on bacterially-expressed dsRNA, knockdown percentages were 80 and 57% knockdown in Acetylcholinesterase transcript, 40 and 60% gene knockdown in Arginine kinase, 74 and 73% knockdown in Chymotrypsin, and 80 and 20% reduction in V-ATPase transcript expression. Overall, the mRNA knockdown percentages in the targeted genes were more pronounced at 5 days of exposure to bacterially-expressed crude dsRNA than 15 days of exposure. However, most purified dsRNAs rarely induce any significant knockdown except dsARG, which reduced the arginine kinase transcript by 40%. Our findings suggest that for optimum RNAi in C. partellus, the dsRNA must be protected from direct access with nucleases. © 2021 Friends Science Publishers

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Identification and validation of potential reference gene for effective dsRNA knockdown analysis in Chilo partellus

Cited by 16 publications

References 48 publications

Identification of Ginger (Zingiber officinale Roscoe) Reference Genes for Gene Expression Analysis

Identification of Ginger (Zingiber officinale Roscoe) Reference Genes for Gene Expression Analysis

Screening and Validation of Reference Genes for RT-qPCR Under Different Honey Bee Viral Infections and dsRNA Treatment

A Protective dsRNA is Crucial for Optimum RNAi Gene Silencing in Chilo partellus

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