<i>In Silico</i> and Validation Approaches for Optimum Conditions of <i>Rattus norvegicus</i> Target Gene qPCR Primers
The qPCR method requires an oligonucleotide pair to prime the amplification process. With the variety of qPCR reagent and primer options available, in silico and laboratory experimental validation approach was needed to validate the most suitable primer for prior use. This article aims to provide in...
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| Format: | Article |
| Language: | English |
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Universitas Gadjah Mada
2023-06-01
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| Series: | Journal of Tropical Biodiversity and Biotechnology |
| Subjects: | |
| Online Access: | https://journal.ugm.ac.id/jtbb/article/view/71765 |
| _version_ | 1827889944182390784 |
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| author | Gracia Alice Victoria Pollo Nyoman Yudi Antara Firman Alamsyah Rarastoeti Pratiwi |
| author_facet | Gracia Alice Victoria Pollo Nyoman Yudi Antara Firman Alamsyah Rarastoeti Pratiwi |
| author_sort | Gracia Alice Victoria Pollo |
| collection | DOAJ |
| description | The qPCR method requires an oligonucleotide pair to prime the amplification process. With the variety of qPCR reagent and primer options available, in silico and laboratory experimental validation approach was needed to validate the most suitable primer for prior use. This article aims to provide in silico analysis of actin alpha-2 smooth muscle (Acta2), fibroblast activation protein (Fap), hypoxanthine phosphoribosyltransferase-1 (Hprt1), platelet-derived growth factor subunit B (Pdgfb), phosphoinositide-3-kinase regulatory subunit-1 (Pik3r1), and vascular cell adhesion molecule-1 (Vcam1) qPCR primer with qPCR and electrophoresis validation. The procedure used in this approach was in silico analysis of primer from published articles and newly designed primer. The analysis was done with Primer-BLAST for gene specificity, Primer-Dimer, OligoCalc for hairpin formation, BLAST Nucleotide for identical sequence screening, and Clustal Omega for product length validation. Experimental validation was done using qPCR for optimal annealing temperature, priming ability, and amplificon specificity, and electrophoresis for product length validation. This assessment resulted in in silico and laboratory experimental validation of Acta2, Fap, Hprt1, Pdgfb, Pik3r1, and Vcam1 primer pairs producing suitable amplicon for qPCR using Rattus norvegicus cDNA with SYBR annealing temperature range of 60-65°C with three mM MgCl2. The primer pair can be used for further qPCR analysis under similar conditions and the procedure stated can be used as starting point for qPCR Primer preparation. |
| first_indexed | 2024-03-12T21:03:07Z |
| format | Article |
| id | doaj.art-8765c1c631f34deda7b3c7f40adb3c0e |
| institution | Directory Open Access Journal |
| issn | 2540-9573 2540-9581 |
| language | English |
| last_indexed | 2024-03-12T21:03:07Z |
| publishDate | 2023-06-01 |
| publisher | Universitas Gadjah Mada |
| record_format | Article |
| series | Journal of Tropical Biodiversity and Biotechnology |
| spelling | doaj.art-8765c1c631f34deda7b3c7f40adb3c0e2023-07-31T02:42:25ZengUniversitas Gadjah MadaJournal of Tropical Biodiversity and Biotechnology2540-95732540-95812023-06-018210.22146/jtbb.7176533913<i>In Silico</i> and Validation Approaches for Optimum Conditions of <i>Rattus norvegicus</i> Target Gene qPCR PrimersGracia Alice Victoria Pollo0Nyoman Yudi Antara1Firman Alamsyah2Rarastoeti Pratiwi3Faculty of Biology, Universitas Gadjah Mada, Yogyakarta, 55281, IndonesiaFaculty of Health, Universitas Kader Bangsa, Palembang, 30129, IndonesiaCenter for Medical Physics and Cancer Research, CTECH Laboratories EDWAR TECHNOLOGY, Tangerang, 15320, IndonesiaFaculty of Biology, Universitas Gadjah Mada, Yogyakarta, 55281, IndonesiaThe qPCR method requires an oligonucleotide pair to prime the amplification process. With the variety of qPCR reagent and primer options available, in silico and laboratory experimental validation approach was needed to validate the most suitable primer for prior use. This article aims to provide in silico analysis of actin alpha-2 smooth muscle (Acta2), fibroblast activation protein (Fap), hypoxanthine phosphoribosyltransferase-1 (Hprt1), platelet-derived growth factor subunit B (Pdgfb), phosphoinositide-3-kinase regulatory subunit-1 (Pik3r1), and vascular cell adhesion molecule-1 (Vcam1) qPCR primer with qPCR and electrophoresis validation. The procedure used in this approach was in silico analysis of primer from published articles and newly designed primer. The analysis was done with Primer-BLAST for gene specificity, Primer-Dimer, OligoCalc for hairpin formation, BLAST Nucleotide for identical sequence screening, and Clustal Omega for product length validation. Experimental validation was done using qPCR for optimal annealing temperature, priming ability, and amplificon specificity, and electrophoresis for product length validation. This assessment resulted in in silico and laboratory experimental validation of Acta2, Fap, Hprt1, Pdgfb, Pik3r1, and Vcam1 primer pairs producing suitable amplicon for qPCR using Rattus norvegicus cDNA with SYBR annealing temperature range of 60-65°C with three mM MgCl2. The primer pair can be used for further qPCR analysis under similar conditions and the procedure stated can be used as starting point for qPCR Primer preparation.https://journal.ugm.ac.id/jtbb/article/view/71765qpcr primerin silicoexperimental validation |
| spellingShingle | Gracia Alice Victoria Pollo Nyoman Yudi Antara Firman Alamsyah Rarastoeti Pratiwi <i>In Silico</i> and Validation Approaches for Optimum Conditions of <i>Rattus norvegicus</i> Target Gene qPCR Primers Journal of Tropical Biodiversity and Biotechnology qpcr primer in silico experimental validation |
| title | <i>In Silico</i> and Validation Approaches for Optimum Conditions of <i>Rattus norvegicus</i> Target Gene qPCR Primers |
| title_full | <i>In Silico</i> and Validation Approaches for Optimum Conditions of <i>Rattus norvegicus</i> Target Gene qPCR Primers |
| title_fullStr | <i>In Silico</i> and Validation Approaches for Optimum Conditions of <i>Rattus norvegicus</i> Target Gene qPCR Primers |
| title_full_unstemmed | <i>In Silico</i> and Validation Approaches for Optimum Conditions of <i>Rattus norvegicus</i> Target Gene qPCR Primers |
| title_short | <i>In Silico</i> and Validation Approaches for Optimum Conditions of <i>Rattus norvegicus</i> Target Gene qPCR Primers |
| title_sort | i in silico i and validation approaches for optimum conditions of i rattus norvegicus i target gene qpcr primers |
| topic | qpcr primer in silico experimental validation |
| url | https://journal.ugm.ac.id/jtbb/article/view/71765 |
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