Sensitive detection of PIK3CA Exon 20 H1047R breast cancer based on lowcost intercalary dye SYBR Green I real-time qPCR assay
Breast cancer is associated with an excessive function of somatic mutation in phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit α (PIK3CA), specifically in exon 9 and 20 hotspots. Exon 20, which contains H1047R, has more potential for an oncogenic mutation than exon 9. Detection of PI...
| Main Authors: | , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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Penerbit Universiti Kebangsaan Malaysia
2024
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| Online Access: | http://journalarticle.ukm.my/24648/1/SS%2012.pdf |
| _version_ | 1824449786413056000 |
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| author | Desriani, Azamris, Rustamadji, Primariadewi Abna, Inherni Marti Ibadurrahman, Irsyad Fuad, Asrul Muhammad Nurdiani, Dini Yuliawati, Yuliawati Herawati, Neng Fitria, Najmiatul Warisman, Muhammad Ali Utami, Nuruliawaty |
| author_facet | Desriani, Azamris, Rustamadji, Primariadewi Abna, Inherni Marti Ibadurrahman, Irsyad Fuad, Asrul Muhammad Nurdiani, Dini Yuliawati, Yuliawati Herawati, Neng Fitria, Najmiatul Warisman, Muhammad Ali Utami, Nuruliawaty |
| author_sort | Desriani, |
| collection | UKM |
| description | Breast cancer is associated with an excessive function of somatic mutation in phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit α (PIK3CA), specifically in exon 9 and 20 hotspots. Exon 20, which contains H1047R, has more potential for an oncogenic mutation than exon 9. Detection of PIK3CA/H1047R mutation has also been reported for molecular diagnosis and therapeutic application. Sensitive methods are required because mutants are mostly present at low levels in a mixture with the wild type. Therefore, this study aimed to explore the development of a low-cost and sensitive PIK3CA exon 20 H1047R detection method using intercalary dye real-time qPCR (quantitative polymerase chain reaction), SYBR Green I. The method was primer design, formulation, specificity, limit of detection, reproducibility, repeatability, and genotyping of 15 DNA (deoxyribonucleic acid)-frozen tissue patient samples, which were further confirmed by PCR sequencing for validation. DNA primer proportion formulations were 0.25 μM PIK3CA exon 20 (WT), 0.65 μM PIK3CA exon 20 H1047 (MT), and 0.2 μM reverse primer in 10 μl total volume. The results showed that compared to the gold standard genotyping PCR sequencing (6.6-20%), the developed method had a higher sensitivity of 5%. The coefficients of intra- and inter-variability were between 0.01-0.19%, suggesting the developed method was repeatable and reproducible with a low mean pipetting error. Genotyping of 15 DNA breast cancer patient samples showed a wild-type genotype, which was in 100% agreement with the PCR sequencing result. The developed method showed sensitive, reproducible, and repeatable results, potentially applicable in prognosis and therapeutic predictions. |
| first_indexed | 2025-02-19T02:08:22Z |
| format | Article |
| id | ukm.eprints-24648 |
| institution | Universiti Kebangsaan Malaysia |
| language | English |
| last_indexed | 2025-02-19T02:08:22Z |
| publishDate | 2024 |
| publisher | Penerbit Universiti Kebangsaan Malaysia |
| record_format | dspace |
| spelling | ukm.eprints-246482025-01-06T04:46:41Z http://journalarticle.ukm.my/24648/ Sensitive detection of PIK3CA Exon 20 H1047R breast cancer based on lowcost intercalary dye SYBR Green I real-time qPCR assay Desriani, Azamris, Rustamadji, Primariadewi Abna, Inherni Marti Ibadurrahman, Irsyad Fuad, Asrul Muhammad Nurdiani, Dini Yuliawati, Yuliawati Herawati, Neng Fitria, Najmiatul Warisman, Muhammad Ali Utami, Nuruliawaty Breast cancer is associated with an excessive function of somatic mutation in phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit α (PIK3CA), specifically in exon 9 and 20 hotspots. Exon 20, which contains H1047R, has more potential for an oncogenic mutation than exon 9. Detection of PIK3CA/H1047R mutation has also been reported for molecular diagnosis and therapeutic application. Sensitive methods are required because mutants are mostly present at low levels in a mixture with the wild type. Therefore, this study aimed to explore the development of a low-cost and sensitive PIK3CA exon 20 H1047R detection method using intercalary dye real-time qPCR (quantitative polymerase chain reaction), SYBR Green I. The method was primer design, formulation, specificity, limit of detection, reproducibility, repeatability, and genotyping of 15 DNA (deoxyribonucleic acid)-frozen tissue patient samples, which were further confirmed by PCR sequencing for validation. DNA primer proportion formulations were 0.25 μM PIK3CA exon 20 (WT), 0.65 μM PIK3CA exon 20 H1047 (MT), and 0.2 μM reverse primer in 10 μl total volume. The results showed that compared to the gold standard genotyping PCR sequencing (6.6-20%), the developed method had a higher sensitivity of 5%. The coefficients of intra- and inter-variability were between 0.01-0.19%, suggesting the developed method was repeatable and reproducible with a low mean pipetting error. Genotyping of 15 DNA breast cancer patient samples showed a wild-type genotype, which was in 100% agreement with the PCR sequencing result. The developed method showed sensitive, reproducible, and repeatable results, potentially applicable in prognosis and therapeutic predictions. Penerbit Universiti Kebangsaan Malaysia 2024 Article PeerReviewed application/pdf en http://journalarticle.ukm.my/24648/1/SS%2012.pdf Desriani, and Azamris, and Rustamadji, Primariadewi and Abna, Inherni Marti and Ibadurrahman, Irsyad and Fuad, Asrul Muhammad and Nurdiani, Dini and Yuliawati, Yuliawati and Herawati, Neng and Fitria, Najmiatul and Warisman, Muhammad Ali and Utami, Nuruliawaty (2024) Sensitive detection of PIK3CA Exon 20 H1047R breast cancer based on lowcost intercalary dye SYBR Green I real-time qPCR assay. Sains Malaysiana, 53 (11). pp. 3683-3693. ISSN 0126-6039 https://www.ukm.my/jsm/english_journals/vol53num11_2024/contentsVol53num11_2024.html |
| spellingShingle | Desriani, Azamris, Rustamadji, Primariadewi Abna, Inherni Marti Ibadurrahman, Irsyad Fuad, Asrul Muhammad Nurdiani, Dini Yuliawati, Yuliawati Herawati, Neng Fitria, Najmiatul Warisman, Muhammad Ali Utami, Nuruliawaty Sensitive detection of PIK3CA Exon 20 H1047R breast cancer based on lowcost intercalary dye SYBR Green I real-time qPCR assay |
| title | Sensitive detection of PIK3CA Exon 20 H1047R breast cancer based on lowcost intercalary dye SYBR Green I real-time qPCR assay |
| title_full | Sensitive detection of PIK3CA Exon 20 H1047R breast cancer based on lowcost intercalary dye SYBR Green I real-time qPCR assay |
| title_fullStr | Sensitive detection of PIK3CA Exon 20 H1047R breast cancer based on lowcost intercalary dye SYBR Green I real-time qPCR assay |
| title_full_unstemmed | Sensitive detection of PIK3CA Exon 20 H1047R breast cancer based on lowcost intercalary dye SYBR Green I real-time qPCR assay |
| title_short | Sensitive detection of PIK3CA Exon 20 H1047R breast cancer based on lowcost intercalary dye SYBR Green I real-time qPCR assay |
| title_sort | sensitive detection of pik3ca exon 20 h1047r breast cancer based on lowcost intercalary dye sybr green i real time qpcr assay |
| url | http://journalarticle.ukm.my/24648/1/SS%2012.pdf |
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