Versatility of different melting temperature (Tm) calculator software for robust PCR and real-time PCR oligonucleotide design: A practical guide

Bakhtiarizadeh, Mohammad Reza; Najafpanah, Mohammad Javad; Mousapour, Hojatollah; Salami, Seyed Alireza

doi:10.1016/j.genrep.2015.11.001

Cited by 8 publications

(7 citation statements)

References 17 publications

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“…First-strand cDNA,s were synthesized using the first strand cDNA synthesis kit (Thermo Fisher, Co., USA) according to the manufacturer’s instructions. Primers were designed using Primer3Plus software 40,41 . The primer sequences are listed in Supplementary File 1.…”

Section: Methodsmentioning

confidence: 99%

Deep transcriptome analysis using RNA-Seq suggests novel insights into molecular aspects of fat-tail metabolism in sheep

Bakhtiarizadeh

Salehi

Alamouti

et al. 2019

Sci Rep

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Fat-tail content of sheep breeds is varied and the molecular mechanisms regulating fat-tail development have not been well characterized. Aiming at better identifying the important candidate genes and their functional pathways contributing to fat deposition in the tail, a comparative transcriptome analysis was performed between fat- (Lori-Bakhtiari) and thin-tailed (Zel) Iranian sheep breeds using RNA-seq. The experiment was conducted on six male lambs (three lambs per each breed) at seven months of age. Four different combinations of aligners and statistical methods including Hisat2 + edgeR, Hisat2 + DESeq2, STAR + edgeR and STAR + DESeq2 were used to identify the differentially expressed genes (DEGs). The DEGs were selected for functional enrichment analysis and protein-protein interaction (PPI) network construction. Module analysis was also conducted to mine the functional sub-networks from the PPI network. In total, 264 genes including 80 up- and 184 down-regulated genes were identified as DEGs. The RNA-Seq results were validated by Q-RT-PCR. Functional analysis of DEGs and the module analysis of PPI network demonstrated that in addition to pathways affecting lipid metabolism, a series of enriched functional terms related to “response to interleukin”, “MAPK signaling pathways”, “Wnt signaling pathway”, “ECM-receptor interaction”, “regulation of actin cytoskeleton”, and “response to cAMP” might contribute to the deposition of fat in tails of sheep. Overall results using RNA-Seq analysis characterized important candidate genes involved in the fatty acid metabolism and regulation of fat deposition, suggesting novel insights into molecular aspects of fat-tail metabolism in sheep. Selected DEGs should be further investigated as potential markers associated with the fat-tail development in sheep breeds.

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Section: Methodsmentioning

confidence: 99%

Deep transcriptome analysis using RNA-Seq suggests novel insights into molecular aspects of fat-tail metabolism in sheep

Bakhtiarizadeh

Salehi

Alamouti

et al. 2019

Sci Rep

Self Cite

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“…The reaction temperature should be low enough to enable binding of the oligonucleotides to the target DNA. However, if the temperature is too low, unspecific binding or secondary structure formation might lead to a reduction of reaction efficiency [53]. Several factors, such as the structure of the oligonucleotides (e.g.…”

Section: Oligonucleotide Designmentioning

confidence: 99%

“…More accurate prediction models of T m were developed under the application of NN parameters, oligonucleotide and salt concentrations [[59], [60], [61], [62], [63], [64], [65], [66]]. Studies have shown that NN based T m prediction models perform best [53,54,65,67].…”

Section: Oligonucleotide Designmentioning

confidence: 99%

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In-silico Design of DNA Oligonucleotides: Challenges and Approaches

Hendling

Barišić

2019

Computational and Structural Biotechnology Journal

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DNA oligonucleotides are essential components of a high number of technologies in molecular biology. The key event of each oligonucleotide-based assay is the specific binding between oligonucleotides and their target DNA. However, single-stranded DNA molecules also tend to bind to unintended targets or themselves. The probability of such unspecific binding increases with the complexity of an assay. Therefore, accurate data management and design workflows are necessary to optimize the in-silico design of primers and probes. Important considerations concerning computational infrastructure and run time need to be made for both data management and the design process. Data retrieval, data updates, storage, filtering and analysis are the main parts of a sequence data management system. Each part needs to be well-implemented as the resulting sequences form the basis for the oligonucleotide design. Important key features, such as the oligonucleotide length, melting temperature, secondary structures and primer dimer formation, as well as the specificity, should be considered for the in-silico selection of oligonucleotides. The development of an efficient oligonucleotide design workflow demands the right balance between the precision of the applied computer models, the general expenditure of time, and computational workload. This paper gives an overview of important parameters during the design process, starting from the data retrieval, up to the design parameters for optimized oligonucleotide design.

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“…Bakhtiarizadeh et al . compared the most common T m calculator tools for PCR oligonucleotide design showing that Primer3 Plus and Primer-BLAST offer the most precise algorithms for an accurate T m prediction ( 6–8 ). The primer pairing parameter needs to consider the size of the PCR product, a minimal T m difference between the primers, or matched Δ G values ( 1 , 4 , 9 ).…”

Section: Introductionmentioning

confidence: 99%

Oli2go: an automated multiplex oligonucleotide design tool

Hendling

Pabinger

Peters

et al. 2018

Nucleic Acids Research

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The success of widely used oligonucleotide-based experiments, ranging from PCR to microarray, strongly depends on an accurate design. The design process involves a number of steps, which use specific parameters to produce high quality oligonucleotides. Oli2go is an efficient, user friendly, fully automated multiplex oligonucleotide design tool, which performs primer and different hybridization probe designs as well as specificity and cross dimer checks in a single run. The main improvement to existing oligonucleotide design web-tools is that oli2go combines multiple steps in an all-in-one solution, where other web applications only accomplish parts of the whole design workflow. Especially, the oli2go specificity check is not only performed against a single species (e.g. mouse), but against bacteria, viruses, fungi, invertebrates, plants, protozoa, archaea and sequences from whole genome shotgun sequence projects and environmental samples, at once. This allows the design of highly specific oligonucleotides in multiplex applications, which is further assured by performing dimer checks not only on the primers themselves, but in an all-against-all fashion. The software is freely accessible to all users at http://oli2go.ait.ac.at/.

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Versatility of different melting temperature (Tm) calculator software for robust PCR and real-time PCR oligonucleotide design: A practical guide

Cited by 8 publications

References 17 publications

Deep transcriptome analysis using RNA-Seq suggests novel insights into molecular aspects of fat-tail metabolism in sheep

Deep transcriptome analysis using RNA-Seq suggests novel insights into molecular aspects of fat-tail metabolism in sheep

In-silico Design of DNA Oligonucleotides: Challenges and Approaches

Oli2go: an automated multiplex oligonucleotide design tool

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