Development of a strand-specific RT-PCR to detect the positive sense replicative strand of Soybean blotchy mosaic virus

Strydom, Elrea; Pietersen, Gerhard

doi:10.1016/j.jviromet.2018.05.014

Cited by 9 publications

(6 citation statements)

References 22 publications

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“…The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/cells13030226/s1, Figure S1: Expression analysis using a peak annotation pipeline; Figure S2: Protein blots for Figure 6B; Figure S3: Mitochondrial dsRNA; Table S1: Primers for dsRNA sensors; Table S2: Statistics of RNA sequencing data. References [3,[59][60][61][62]…”

Section: Supplementary Materialsmentioning

confidence: 99%

Significant Variations in Double-Stranded RNA Levels in Cultured Skin Cells

Sadeq,

Chitcharoen,

Al-Hashimi

et al. 2024

Cells

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Endogenous double-stranded RNA has emerged as a potent stimulator of innate immunity. Under physiological conditions, endogenous dsRNA is maintained in the cell nucleus or the mitochondria; however, if protective mechanisms are breached, it leaches into the cytoplasm and triggers immune signaling pathways. Ectopic activation of innate immune pathways is associated with various diseases and senescence and can trigger apoptosis. Hereby, the level of cytoplasmic dsRNA is crucial. We have enriched dsRNA from two melanoma cell lines and primary dermal fibroblasts, including a competing probe, and analyzed the dsRNA transcriptome using RNA sequencing. There was a striking difference in read counts between the cell lines and the primary cells, and the effect was confirmed by northern blotting and immunocytochemistry. Both mitochondria (10–20%) and nuclear transcription (80–90%) contributed significantly to the dsRNA transcriptome. The mitochondrial contribution was lower in the cancer cells compared to fibroblasts. The expression of different transposable element families was comparable, suggesting a general up-regulation of transposable element expression rather than stimulation of a specific sub-family. Sequencing of the input control revealed minor differences in dsRNA processing pathways with an upregulation of oligoadenylate synthase and RNP125 that negatively regulates the dsRNA sensors RIG1 and MDA5. Moreover, RT-qPCR, Western blotting, and immunocytochemistry confirmed the relatively minor adaptations to the hugely different dsRNA levels. As a consequence, these transformed cell lines are potentially less tolerant to interventions that increase the formation of endogenous dsRNA.

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Section: Supplementary Materialsmentioning

confidence: 99%

Significant Variations in Double-Stranded RNA Levels in Cultured Skin Cells

Sadeq,

Chitcharoen,

Al-Hashimi

et al. 2024

Cells

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“…Negative-sense CiLV-C viral genomes detected in mite extracts ( Roy et al, 2015 ) could have been remnants from the infected plant cells after mite feeding ( Tassi et al, 2017 ). Moreover, the specific detection of the viral negative-strand RNA by reverse transcription-polymerase chain reaction (RT-PCR) could have been the result of false amplification due to either self-priming of the positive-strand RNA or the primer activity of other cellular nucleic acids ( Haddad et al, 2007 ; Boncristiani et al, 2009 ; Haist et al, 2015 ; Strydom and Pietersen, 2018 ).…”

Section: Introductionmentioning

confidence: 99%

Molecular Epidemiology of Citrus Leprosis Virus C: A New Viral Lineage and Phylodynamic of the Main Viral Subpopulations in the Americas

et al. 2021

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Despite the importance of viral strains/variants as agents of emerging diseases, genetic and evolutionary processes affecting their ecology are not fully understood. To get insight into this topic, we assessed the population and spatial dynamic parameters of citrus leprosis virus C (CiLV-C, genus Cilevirus, family Kitaviridae). CiLV-C is the etiological agent of citrus leprosis disease, a non-systemic infection considered the main viral disorder affecting citrus orchards in Brazil. Overall, we obtained 18 complete or near-complete viral genomes, 123 complete nucleotide sequences of the open reading frame (ORF) encoding the putative coat protein, and 204 partial nucleotide sequences of the ORF encoding the movement protein, from 430 infected Citrus spp. samples collected between 1932 and 2020. A thorough examination of the collected dataset suggested that the CiLV-C population consists of the major lineages CRD and SJP, unevenly distributed, plus a third one called ASU identified in this work, which is represented by a single isolate found in an herbarium sample collected in Asuncion, Paraguay, in 1937. Viruses from the three lineages share about 85% nucleotide sequence identity and show signs of inter-clade recombination events. Members of the lineage CRD were identified both in commercial and non-commercial citrus orchards. However, those of the lineages SJP were exclusively detected in samples collected in the citrus belt of São Paulo and Minas Gerais, the leading Brazilian citrus production region, after 2015. The most recent common ancestor of viruses of the three lineages dates back to, at least, ∼1500 years ago. Since citrus plants were introduced in the Americas by the Portuguese around the 1520s, the Bayesian phylodynamic analysis suggested that the ancestors of the main CiLV-C lineages likely originated in contact with native vegetation of South America. The intensive expansion of CRD and SJP lineages in Brazil started probably linked to the beginning of the local citrus industry. The high prevalence of CiLV-C in the citrus belt of Brazil likely ensues from the intensive connectivity between orchards, which represents a potential risk toward pathogen saturation across the region.

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“…It is generally assumed that priming RT with gene-specific primer (GSP) for a particular transcript would generate cDNA only from that desired RNA template and guarantee the specificity of signal in subsequent amplification by PCR. However, the conventional notion regarding absolute requirement of the exogenous primers during RT has been refuted by several reports involving detection of plant [1,2], animal [3][4][5] and human viruses [6,7] as well as expression of genes in eukaryotes [6][7][8][9], in which it was observed that cDNAs can be synthesized even without adding exogenous primers to the RT reaction. Generation of such nonspecific cDNAs through a primer independent mechanism has been linked to self-priming activity of the RNA template by its own 3 end [6] and previously termed as background priming [4,8].…”

Section: Introductionmentioning

confidence: 99%

“…To circumvent the problem of background priming in RT-PCR, a multitude of approaches have been adopted at various stages, such as additional processing of sample RNA, variations at the level of cDNA synthesis and PCR amplification after RT [1][2][3][5][6][7][8][9][10][11][12]. These strategies include elimination of free nucleic acid contaminants from the RNA sample that can act as endogenous primers, RNase-Hmediated digestion of nontarget RNA strand, performing RT at a higher extension temperature, addition of DMSO to RT reaction, use of a thermostable reverse transcriptase, using reverse transcriptase with RNase-H activity and using one-step RT-PCR kits.…”

Section: Introductionmentioning

confidence: 99%

“…Furthermore, the incorporation of additional tags in RT primers and subsequent detection of tagged cDNAs, modifying the sequence of primer-specific cDNA, digestion of unused RT primers with Exonuclease-I and purification of cDNA synthesized with biotinylated primers have also been tested in several methods. Although these strategies when applied alone did not improve the strand-specific detection of cDNAs [1,3,[6][7][8]12], few combinatorial approaches have shown a varying degree of success [2,5,[9][10][11].…”

Section: Introductionmentioning

confidence: 99%

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Strand-Specific Detection of Overlapping Transcripts Via Purification Involving Denaturation of Biotinylated cDNA

Uddin

Srivastava

2020

BioTechniques

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Reverse transcription-PCR (RT-PCR) is the most widely employed technique for gene expression analysis owing to its high sensitivity, easy reproducibility and fast output. It has been conceived that priming RT reactions with gene-specific primers generates cDNA only from the specific RNA. However, several reports have revealed that cDNA is synthesized even without addition of exogenous primers in RT reactions. Owing to such self-priming activity, the signals from specific strands cannot be accurately detected and can confound the expression analysis, especially in context of overlapping bidirectional transcripts. Here, we demonstrate that purification of biotin-tagged cDNA in conjunction with alkaline denaturation can obviate the problem of background priming and enable accurate strand-specific detection of overlapping transcripts.

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Development of a strand-specific RT-PCR to detect the positive sense replicative strand of Soybean blotchy mosaic virus

Cited by 9 publications

References 22 publications

Significant Variations in Double-Stranded RNA Levels in Cultured Skin Cells

Significant Variations in Double-Stranded RNA Levels in Cultured Skin Cells

Molecular Epidemiology of Citrus Leprosis Virus C: A New Viral Lineage and Phylodynamic of the Main Viral Subpopulations in the Americas

Strand-Specific Detection of Overlapping Transcripts Via Purification Involving Denaturation of Biotinylated cDNA

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