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In Silico and Validation Approaches for Optimum Conditions of Rattus norvegicus Target Gene qPCR Primers

Journal of Tropical Biodiversity and Biotechnology · Vol. 8 No. 2 (2023)

DOI
10.22146/jtbb.71765
ISSN
2540-9581, 2540-9573
Terbit
2023-06-05
Penerbit
Universitas Gadjah Mada
Bahasa
eng
Lisensi
creativecommons.org/licenses/by-sa/4.0

Abstrak

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.

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  • Universitas Gadjah Mada
  • Universitas Al-Azhar Indonesia
  • National Research and Innovation Agency
  • Andalas University
  • Padjadjaran University
  • Universitas Jenderal Achmad Yani
  • Universitas Halu Oleo
  • Organisasi lain
  • Afiliasi tidak diketahui
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Rekan penulisOrganisasiArtikel bersama
Warsito Purwo TarunoAfiliasi tidak diketahui4
M. MurwantokoAfiliasi tidak diketahui3
Rumiyati RumiyatiUniversitas Gadjah Mada3
PUDJI ASTUTIAfiliasi tidak diketahui3
Alfi Rizqi AmaliaUniversitas Gadjah Mada2
Woro Anindito Sri TunjungUniversitas Gadjah Mada2
Yekti Asih PurwestriUniversitas Gadjah Mada2
Nita NorikoUniversitas Al-Azhar Indonesia1
Arief PambudiUniversitas Al-Azhar Indonesia1
Siti Mimah RohimahUniversitas Al-Azhar Indonesia1
Dewi ElfidasariUniversitas Al-Azhar Indonesia1
Rizky Nuur Berlianni MulyantoUniversitas Al-Azhar Indonesia1
Rita MalizaAndalas University1
Putra SantosoAndalas University1
Bramadi AryaMahidol University1
Alimuddin TofrizalAndalas University1
Wahyu WidowatiMaranatha Christian University1
Ervi AfifahAfiliasi tidak diketahui1
Rizal RizalUniversity of Indonesia1
Omat Rachmat HasbullahSultan Ageng Tirtayasa University1
Ilma FiddiyantiUniversitas Jenderal Achmad Yani1
Dewi Ratih HandayaniUniversitas Jenderal Achmad Yani1
Endang SutedjaPadjadjaran University1
Darmadji IsmonoPadjadjaran University1
Nucki Nursjamsi HidajatPadjadjaran University1
Hanna Sari Widya KusumaAfiliasi tidak diketahui1
Suwarno HadisusantoUniversitas Gadjah Mada1
Sari Budi Moria SembiringNational Research and Innovation Agency1
Jhon Harianto HutapeaNational Research and Innovation Agency1
I Nyoman Adiasmara GiriNational Research and Innovation Agency1
Ketut MahardikaNational Research and Innovation Agency1
HaryantiNational Research and Innovation Agency1
Christina Retna HandayaniMinistry of Marine Affairs and Fisheries1
Asmarani KusumawatiAfiliasi tidak diketahui1
Penny Humaidah HamidAfiliasi tidak diketahui1
Dewi Nur’aeni SetyowatiUniversity of Mataram1

Afiliasi berasal dari OpenAlex; metadata OAI-PMH jurnal tidak memuatnya. Penulis tanpa ID OpenAlex dicocokkan berdasarkan nama, sehingga dua orang bisa tergabung atau satu orang bisa terpisah.

Sumber metadata: OAI-PMH (metadata jurnal) https://jurnal.ugm.ac.id/jtbb/oai, pertama di-harvest 9 Nov 2026, terakhir terlihat 9 Nov 2026.
Metadata asli (oai_dc) 
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	<dc:title xml:lang="en-US">In Silico and Validation Approaches for Optimum Conditions of Rattus norvegicus Target Gene qPCR Primers</dc:title>
	<dc:creator>Pollo, Gracia Alice Victoria</dc:creator>
	<dc:creator>Antara, Nyoman Yudi</dc:creator>
	<dc:creator>Alamsyah, Firman</dc:creator>
	<dc:creator>Pratiwi, Rarastoeti</dc:creator>
	<dc:subject xml:lang="en-US">Biology; Bioinformatics</dc:subject>
	<dc:subject xml:lang="en-US">qPCR primer; in silico; experimental validation</dc:subject>
	<dc:description xml:lang="en-US">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.</dc:description>
	<dc:publisher xml:lang="en-US">Universitas Gadjah Mada</dc:publisher>
	<dc:contributor xml:lang="en-US"></dc:contributor>
	<dc:date>2023-06-05</dc:date>
	<dc:type>info:eu-repo/semantics/article</dc:type>
	<dc:type>info:eu-repo/semantics/publishedVersion</dc:type>
	<dc:type xml:lang="en-US"></dc:type>
	<dc:format>application/pdf</dc:format>
	<dc:identifier>https://journal.ugm.ac.id/jtbb/article/view/71765</dc:identifier>
	<dc:identifier>10.22146/jtbb.71765</dc:identifier>
	<dc:source xml:lang="en-US">Journal of Tropical Biodiversity and Biotechnology; Vol 8, No 2 (2023): August; jtbb71765</dc:source>
	<dc:source>2540-9581</dc:source>
	<dc:source>2540-9573</dc:source>
	<dc:language>eng</dc:language>
	<dc:relation>https://journal.ugm.ac.id/jtbb/article/view/71765/36118</dc:relation>
	<dc:relation>https://journal.ugm.ac.id/jtbb/article/downloadSuppFile/71765/19821</dc:relation>
	<dc:relation>https://journal.ugm.ac.id/jtbb/article/downloadSuppFile/71765/19822</dc:relation>
	<dc:relation>https://journal.ugm.ac.id/jtbb/article/downloadSuppFile/71765/19823</dc:relation>
	<dc:coverage xml:lang="en-US">South East Asia</dc:coverage>
	<dc:coverage xml:lang="en-US">21st century</dc:coverage>
	<dc:coverage xml:lang="en-US">Synthesized DNA oligonucleotides</dc:coverage>
	<dc:rights xml:lang="en-US">Copyright (c) 2023 Journal of Tropical Biodiversity and Biotechnology</dc:rights>
	<dc:rights xml:lang="en-US">http://creativecommons.org/licenses/by-sa/4.0</dc:rights>
</oai_dc:dc>