High-Throughput Approaches for MicroRNA Expression Analysis

Dedeoğlu, Bala Gür

doi:10.1007/978-1-62703-748-8_6

Cited by 23 publications

(20 citation statements)

References 58 publications

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“…Quantitative real-time PCR was performed on the ABI Prism 7300 HT Sequence Detection system (Applied Biosystems, Forster City, CA) using Power SYBR Green PCR Master Mix (Applied Biosystems) and the expression of miRNA was measured through Stem-loop RT-PCR method (Dedeoglu 2014). The relative expression level of CpCPR5 was normalized to the internal control b-actin, and U6 small nuclear (U6) was used to normalize the expression level of miR-932.…”

Section: Quantitative Rt-pcr Analysismentioning

confidence: 99%

MiR-932 Regulates Pyrethroid Resistance inCulex pipiens pallens(Diptera: Culicidae)

Liu¹,

Tian²,

Guo³

et al. 2016

J Med Entomol

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MicroRNAs (miRNAs) play notable role in regulation of gene expression at the posttranscription level, and have been involved in many biological processes, including insecticide resistance. In this study, we investigated the role of miR-932 in the molecular mechanisms of pyrethroid resistance in Culex pipiens pallens (L.). Overexpression of miR-932 in the DS-strain made the mosquitoes more resistant to deltamethrin, while inhibiting the expression of miR-932 in the DR-strain made the mosquitoes more sensitive to deltamethrin. Further, we also identified CpCPR5 as a target gene of miR-932. Sustained overexpression of miR-932 resulted in repression of CpCPR5, and that knockdown of miR-932 increased CpCPR5 expression. In addition, knockdown of CpCPR5 decreased the sensitivity of mosquitoes to deltamethrin in the DS-strain. In conclusion, our study finds a molecular link between miR-932 and CpCPR5 and provides a novel insight into the mechanism of insecticide resistance.Key words: Culex pipiens pallens, pyrethroid, microRNA, CpCPR5Mosquitoes transmit a lot of pathogens, causing severe diseases such as malaria, dengue fever, West Nile fever, encephalitis, and filariasis, which threat public health worldwide (Andriessen et al. 2015, Fortuna et al. 2015, Paredes-Esquivel et al. 2015. Recent estimates show that there are about 400 million infections of dengue fever annually around the world (Sood et al. 2015). According to the WHO world malaria report, there are still 214 million new infections worldwide in 2015. Therefore, it is imperative for controlling and preventing mosquito-borne diseases. However, due to the lack of effective vaccines and drugs, vector control mostly relies on insecticides to reduce the number of mosquitoes (Glunt et al. 2015). But continuous and lasting application of insecticides has led to a constant increasing resistance, which makes antivector strategies more challenging (Rasic et al. 2015). Insecticide resistance arises from complex polygenic inheritance and many genes involved , Bonizzoni et al. 2015. These genes can be regulated at transcriptional and posttranscriptional levels and microRNA (miRNA) is known to be a posttranscriptional gene regulator (Wang et al. 2016).MiRNA is a class of small noncoding RNA, with about 22 nucleotides in length. It has been shown to repress translation via imperfectly pairing with complementary sites within the 3'UTR of target mRNA (Liu et al. 2014, Munoz et al. 2015. In diverse cellular programs, miRNA plays a key role and involves in cell growth, differentiation, apoptosis, and many other physiological processes , Park et al. 2015. In insects, the studies on miRNAs are mostly focused on the development, behavior, and hostpathogen interactions (Lucas et al. 2015). Recent evidence has shown that a few miRNAs participate in the regulation of insecticide resistance in mosquitoes, but the exact role of miRNA in pyrethroid resistance is still under little attention yet .Here, according to our previous study, we identified many different expressed miRNAs...

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Section: Quantitative Rt-pcr Analysismentioning

confidence: 99%

MiR-932 Regulates Pyrethroid Resistance inCulex pipiens pallens(Diptera: Culicidae)

Liu¹,

Tian²,

Guo³

et al. 2016

J Med Entomol

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“…The results demonstrated that there was a general consistency between qPCR and high-throughput sequencing, though there were some differences in the results of the two different technologies. Previous studies have indicated that RNA-seq is an accurate and powerful tool to identify new miRNAs and quantify miRNA expression [25,26]. On the other hand, the small size and heterogeneity of microRNAs present challenges for detection by RT-qPCR.…”

Section: Resultsmentioning

confidence: 99%

MicroRNA Transcriptome of Poly I:C-Stimulated Peripheral Blood Mononuclear Cells Reveals Evidence for MicroRNAs in Regulating Host Response to RNA Viruses in Pigs

Wang

et al. 2016

IJMS

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MicroRNAs (miRNAs) are one family of small noncoding RNAs that function to modulate the activity of specific mRNA targets in animals. To understand the role of miRNAs in regulating genes involved in the host immune response to RNA viruses, we profiled and characterized the miRNAs of swine peripheral blood mononuclear cells (PBMC) stimulated with poly I:C, a synthetic dsRNA analog, by miRNA-sequencing (miRNA-seq). We identified a total of 905 miRNAs, of which 503 miRNAs were firstly exploited herein with no annotation in the latest miRBase 21.0. Expression analysis demonstrated that poly I:C stimulation can elicit significantly differentially expressed (DE) miRNAs in Dapulian (n = 20), one Chinese indigenous breed, as well as Landrace (n = 23). By integrating the mRNA expression profiles of the same sample with miRNA profiles, we carried out function analyses of the target genes of these DE miRNAs, with the results indicating that target genes were most enriched in some immune-related pathways and gene ontology (GO) terms, suggesting that DE miRNAs play an important role in the regulation of host to poly I:C stimulation. Furthermore, we also detected 43 and 61 significantly DE miRNAs between the two breeds in the control sample groups and poly I:C stimulation groups, respectively, which may be involved in regulation of the different characteristics of the two breeds. This study describes for the first time the PBMC miRNA transcriptomic response to poly I:C stimulation in pigs, which not only contributes to a broad view of the pig miRNAome but improves our understanding of miRNA function in regulating host immune response to RNA viruses.

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“…The sequences of primer for CYP6AG11 were listed in Table 1. MiRNA quantification was calculated by the Bulge-loop™ miRNA qRT-PCR method (Dedeoglu 2014; Fulci et al 2007) and primer sets (one RT primer and a pair of qPCR primers) specific for miR-278-3p were designed and listed in Table 1. The relative expression level of CYP6AG11 was normalized to the internal control β-actin , and U6 small nuclear ( U6 ) was used to normalize the expression level of miR-278-3p.…”

Section: Methodsmentioning

confidence: 99%

MiR-278-3p regulates pyrethroid resistance in Culex pipiens pallens

Lei

Wang

et al. 2014

Parasitol Res

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MicroRNAs (miRNAs) regulate gene expression and biological processes including embryonic development, innate immunity and infection in many species. Emerging evidence indicates that miRNAs are involved in drug resistance. However, little is known about the relationship between the miRNAs and insecticide resistance in mosquitos. Here, we reported that conserved miR-278-3p and its target gene are critical for pyrethroid resistance in Culex pipiens pallens. We found that CYP6AG11 is the target of miR-278-3p, through bioinformatic analysis and experimental verification. The expression level of miR-278-3p was lower, whereas the level of CYP6AG11 was higher in deltamethrin-resistant strain, which were detected using qRT-PCR. We also found that CYP6AG11 was regulated by miR-278-3p via a specific target site with the 3′UTR by luciferase reporter assay. In addition, overexpression of CYP6AG11 in the mosquito C6/36 cells showed better prolification than the cells with empty vector when treated by deltamethrin at different concentrations. Moreover, the overexpression of miR-278-3p through microinjection led to a significant reduction in the survival rate, and the level of CYP6AG11 was simultaneously reduced. These results indicated that miR-278-3p could regulate the pyrethroid resistance through CYP6AG11.

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High-Throughput Approaches for MicroRNA Expression Analysis

Cited by 23 publications

References 58 publications

MiR-932 Regulates Pyrethroid Resistance inCulex pipiens pallens(Diptera: Culicidae)

MiR-932 Regulates Pyrethroid Resistance inCulex pipiens pallens(Diptera: Culicidae)

MicroRNA Transcriptome of Poly I:C-Stimulated Peripheral Blood Mononuclear Cells Reveals Evidence for MicroRNAs in Regulating Host Response to RNA Viruses in Pigs

MiR-278-3p regulates pyrethroid resistance in Culex pipiens pallens

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