Ex-Ex Primer: An experimentally validated tool for designing oligonucleotides spanning spliced nucleic acid regions from multiple species

Govindkumar, Balagannavar; Basavaraju, Kavyashree; Patel, Krishna; Sasidharan, Kalesh; Arumugam, T. Siva; Thomas, Lijo; Praveena, B.K.G.; Raksha, H.N.; Menon, Rajasree; Acharya, Kshitish K.

doi:10.1016/j.jbiotec.2021.10.009

Cited by 5 publications

(3 citation statements)

References 16 publications

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“…The primer set used for qPCR was tested using the basic local alignment search tool (BLAST, https://blast.ncbi.nlm.nih.gov/Blast.cgi ) against both the NCBI database and internally in our unfiltered long-read transcriptomic data to provide specificity (‘virtual PCR’ approach, following published principles [ 53 ]). Quality control of the primers after qPCR was also performed by obtaining and evaluating the melting plots for every primer pair as well as gel electrophoresis of amplificated cDNA fragments to check for estimated and real fragment length, followed by Sanger sequencing of the band to proof sequence identity.…”

Section: Methodsmentioning

confidence: 99%

Full-length transcriptomic analysis in murine and human heart reveals diversity of PGC-1α promoters and isoforms regulated distinctly in myocardial ischemia and obesity

et al. 2022

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Background Peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α) acts as a transcriptional coactivator and regulates mitochondrial function. Various isoforms are generated by alternative splicing and differentially regulated promoters. In the heart, total PGC-1α deficiency knockout leads to dilatative cardiomyopathy, but knowledge on the complexity of cardiac isoform expression of PGC-1α remains sparse. Thus, this study aims to generate a reliable dataset on cardiac isoform expression pattern by long-read mRNA sequencing, followed by investigation of differential regulation of PGC-1α isoforms under metabolic and ischemic stress, using high-fat-high-sucrose-diet-induced obesity and a murine model of myocardial infarction. Results Murine (C57Bl/6J) or human heart tissue (obtained during LVAD-surgery) was used for long-read mRNA sequencing, resulting in full-length transcriptomes including 58,000 mRNA isoforms with 99% sequence accuracy. Automatic bioinformatic analysis as well as manual similarity search against exonic sequences leads to identification of putative coding PGC-1α isoforms, validated by PCR and Sanger sequencing. Thereby, 12 novel transcripts generated by hitherto unknown splicing events were detected. In addition, we postulate a novel promoter with homologous and strongly conserved sequence in human heart. High-fat diet as well as ischemia/reperfusion (I/R) injury transiently reduced cardiac expression of PGC-1α isoforms, with the most pronounced effect in the infarcted area. Recovery of PGC-1α-isoform expression was even more decelerated when I/R was performed in diet-induced obese mice. Conclusions We deciphered for the first time a complete full-length transcriptome of the murine and human heart, identifying novel putative PGC-1α coding transcripts including a novel promoter. These transcripts are differentially regulated in I/R and obesity suggesting transcriptional regulation and alternative splicing that may modulate PGC-1α function in the injured and metabolically challenged heart.

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Section: Methodsmentioning

confidence: 99%

Full-length transcriptomic analysis in murine and human heart reveals diversity of PGC-1α promoters and isoforms regulated distinctly in myocardial ischemia and obesity

et al. 2022

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“…We selected another control gene from the list of un-differential genes showing the highest p value and lowest logFC across all treatment conditions. Primers for the genes were designed using the Ex-Ex Primer tool 39 . A list of primers used is provided in Supplementary file 1.…”

Section: Methodsmentioning

confidence: 99%

Transcriptome analysis of Sparidentex hasta larvae exposed to water-accommodated fraction of Kuwait crude oil

Kumar,

Karam,

Shajan

et al. 2024

Sci Rep

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Anthropogenic activities have been shown to significantly affect marine life. Water pollution and oil spills are particularly deleterious to the fish population, especially during their larval stage. In this study, Sobaity-sea bream Sparidentex hasta (Valenciennes, 1830) larvae were exposed to serial dilutions of water-accommodated fraction of Kuwait crude oil (KCO-WAF) for varying durations (3, 6, 24, 48, 72 or 96 h) in acute exposure regime. Gene expression was assessed using RNA sequencing and validated through RT-qPCR. The RNA sequencing data were aligned to the sequenced genome, and differentially expressed genes were identified in response to treatment with or without KCO-WAF at various exposure times. The highest number of differentially expressed genes was observed at the early time point of 6 h of post-exposure to KCO-WAF. The lowest number of differentially expressed genes were noticed at 96 h of treatment indicating early response of the larvae to KCO-WAF contaminant. The acquired information on the differentially expressed genes was then used for functional and pathway analysis. More than 90% of the differentially expressed genes had a significant BLAST match, with the two most common matching species being Acanthopagrus latus and Sparus aurata. Approximately 65% of the differentially expressed genes had Gene Ontology annotations, whereas > 35% of the genes had KEGG pathway annotations. The differentially expressed genes were found to be enriched for various signaling pathways (e.g., MAPK, cAMP, PI3K-Akt) and nervous system-related pathways (e.g., neurodegeneration, axon guidance, glutamatergic synapse, GABAergic synapse). Early exposure modulated the signaling pathways, while KCO-WAF exposure of larvae for a longer duration affected the neurodegenerative/nervous system-related pathways. RT-qPCR analysis confirmed the differential expression of genes at each time point. These findings provide insights into the underlying molecular mechanisms of the deleterious effects of acute exposure to oil pollution—on marine fish populations, particularly at the early larval stage of Sparidentex hasta.

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“…In addition, it presumably leverages Primer3’s built-in capability to detect dimers or hairpins, and there is extensive room for improvement in this area [ 8 ]. Other more RT-qPCR-focused tools include Ex-Ex-Primer [ 9 ] QuantPrimer [ 10 ], IDT’s private software ( https://eu.idtdna.com/scitools/Applications/RealTimePCR ) and the highly versatile FastPCR [ 11 ]. While Ex-Ex-Primer assists a wide variety of qPCR setups, it requires the user to input the target junction, thus entailing the need for a prior multiple sequence alignment in the case of genes with several transcripts.…”

Section: Introductionmentioning

confidence: 99%

ExonSurfer: a web-tool to design primers at exon–exon junctions

Monfort-Lanzas,

Rusu,

Parrakova

et al. 2024

BMC Genomics

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Background Reverse transcription quantitative PCR (RT-qPCR) with intercalating dyes is one of the main techniques to assess gene expression levels used in basic and applied research as well as in diagnostics. However, primer design for RT-qPCR can be complex due to the high demands on primer quality. Primers are best placed on exon junctions, should avoid polymorphic regions, be specific to the target transcripts and also prevent genomic amplification accurately, among others. Current software tools manage to meet all the necessary criteria only insufficiently. Here, we present ExonSurfer, a novel, user-friendly web-tool for qPCR primer design. Results ExonSurfer combines the different steps of the primer design process, encompassing target selection, specificity and self-complementarity assessment, and the avoidance of issues arising from polymorphisms. Amplification of potentially contaminating genomic DNA is avoided by designing primers on exon-exon junctions, moreover, a genomic alignment is performed to filter the primers accordingly and inform the user of any predicted interaction. In order to test the whole performance of the application, we designed primer pairs for 26 targets and checked both primer efficiency, amplicon melting temperature and length and confirmed the targeted amplicon by Sanger sequencing. Most of the tested primers accurately and selectively amplified the corresponding targets. Conclusion ExonSurfer offers a comprehensive end-to-end primer design, guaranteeing transcript-specific amplification. The user interface is intuitive, providing essential specificity and amplicon details. The tool can also be used by command line and the source code is available. Overall, we expect ExonSurfer to facilitate RT-qPCR set-up for researchers in many fields.

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Ex-Ex Primer: An experimentally validated tool for designing oligonucleotides spanning spliced nucleic acid regions from multiple species

Cited by 5 publications

References 16 publications

Full-length transcriptomic analysis in murine and human heart reveals diversity of PGC-1α promoters and isoforms regulated distinctly in myocardial ischemia and obesity

Full-length transcriptomic analysis in murine and human heart reveals diversity of PGC-1α promoters and isoforms regulated distinctly in myocardial ischemia and obesity

Transcriptome analysis of Sparidentex hasta larvae exposed to water-accommodated fraction of Kuwait crude oil

ExonSurfer: a web-tool to design primers at exon–exon junctions

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