Optimized RT-qPCR and a novel normalization method for validating circulating miRNA biomarkers in ageing-related diseases
Abstract Circulating miRNAs have potential as minimally invasive biomarkers for diagnosing various diseases, including ageing-related disorders such as Alzheimer’s disease (AD). However, the lack of standardization in the common analysis method, RT-qPCR, and specifically in the normalization step, h...
| Main Authors: | , , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Nature Portfolio
2023-11-01
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| Series: | Scientific Reports |
| Online Access: | https://doi.org/10.1038/s41598-023-47971-3 |
| _version_ | 1797415564622168064 |
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| author | Andrew Want Karolina Staniak Wioleta Grabowska-Pyrzewicz Aleksandra Fesiuk Anna Barczak Tomasz Gabryelewicz Agnieszka Kulczyńska-Przybik Barbara Mroczko Urszula Wojda |
| author_facet | Andrew Want Karolina Staniak Wioleta Grabowska-Pyrzewicz Aleksandra Fesiuk Anna Barczak Tomasz Gabryelewicz Agnieszka Kulczyńska-Przybik Barbara Mroczko Urszula Wojda |
| author_sort | Andrew Want |
| collection | DOAJ |
| description | Abstract Circulating miRNAs have potential as minimally invasive biomarkers for diagnosing various diseases, including ageing-related disorders such as Alzheimer’s disease (AD). However, the lack of standardization in the common analysis method, RT-qPCR, and specifically in the normalization step, has resulted in inconsistent data across studies, hindering miRNA clinical implementation as well as basic research. To address this issue, this study proposes an optimized protocol for key steps in miRNA profiling, which incorporates absorbance-based haemolysis detection for assessing sample quality, double spike-in controls for miRNA isolation and reverse transcription, and the use of 7 stable normalizers verified in an aging population, including healthy subjects and individuals at different stages of Alzheimer's disease (140 subjects). The stability of these 7 normalizers was demonstrated using our novel method called BestmiRNorm for identifying optimal normalizers. BestmiRNorm, developed utilizing the Python programming language, enables the assessment of up to 11 potential normalizers. The standardized application of this optimized RT-qPCR protocol and the recommended normalizers are crucial for the development of miRNAs as biomarkers for AD and other ageing-related diseases in clinical diagnostics and basic research. |
| first_indexed | 2024-03-09T05:49:24Z |
| format | Article |
| id | doaj.art-aee6d7077508426092283d6fd5bba37a |
| institution | Directory Open Access Journal |
| issn | 2045-2322 |
| language | English |
| last_indexed | 2024-03-09T05:49:24Z |
| publishDate | 2023-11-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Scientific Reports |
| spelling | doaj.art-aee6d7077508426092283d6fd5bba37a2023-12-03T12:18:19ZengNature PortfolioScientific Reports2045-23222023-11-0113111310.1038/s41598-023-47971-3Optimized RT-qPCR and a novel normalization method for validating circulating miRNA biomarkers in ageing-related diseasesAndrew Want0Karolina Staniak1Wioleta Grabowska-Pyrzewicz2Aleksandra Fesiuk3Anna Barczak4Tomasz Gabryelewicz5Agnieszka Kulczyńska-Przybik6Barbara Mroczko7Urszula Wojda8Laboratory of Preclinical Testing of Higher Standard, Nencki Institute of Experimental Biology, Polish Academy of SciencesLaboratory of Preclinical Testing of Higher Standard, Nencki Institute of Experimental Biology, Polish Academy of SciencesLaboratory of Preclinical Testing of Higher Standard, Nencki Institute of Experimental Biology, Polish Academy of SciencesLaboratory of Preclinical Testing of Higher Standard, Nencki Institute of Experimental Biology, Polish Academy of SciencesDementia Diseases Unit, Mossakowski Medical Research Institute, Polish Academy of SciencesDementia Diseases Unit, Mossakowski Medical Research Institute, Polish Academy of SciencesDepartment of Neurodegeneration Diagnostics, Faculty of Medicine, Medical University of BialystokDepartment of Neurodegeneration Diagnostics, Faculty of Medicine, Medical University of BialystokLaboratory of Preclinical Testing of Higher Standard, Nencki Institute of Experimental Biology, Polish Academy of SciencesAbstract Circulating miRNAs have potential as minimally invasive biomarkers for diagnosing various diseases, including ageing-related disorders such as Alzheimer’s disease (AD). However, the lack of standardization in the common analysis method, RT-qPCR, and specifically in the normalization step, has resulted in inconsistent data across studies, hindering miRNA clinical implementation as well as basic research. To address this issue, this study proposes an optimized protocol for key steps in miRNA profiling, which incorporates absorbance-based haemolysis detection for assessing sample quality, double spike-in controls for miRNA isolation and reverse transcription, and the use of 7 stable normalizers verified in an aging population, including healthy subjects and individuals at different stages of Alzheimer's disease (140 subjects). The stability of these 7 normalizers was demonstrated using our novel method called BestmiRNorm for identifying optimal normalizers. BestmiRNorm, developed utilizing the Python programming language, enables the assessment of up to 11 potential normalizers. The standardized application of this optimized RT-qPCR protocol and the recommended normalizers are crucial for the development of miRNAs as biomarkers for AD and other ageing-related diseases in clinical diagnostics and basic research.https://doi.org/10.1038/s41598-023-47971-3 |
| spellingShingle | Andrew Want Karolina Staniak Wioleta Grabowska-Pyrzewicz Aleksandra Fesiuk Anna Barczak Tomasz Gabryelewicz Agnieszka Kulczyńska-Przybik Barbara Mroczko Urszula Wojda Optimized RT-qPCR and a novel normalization method for validating circulating miRNA biomarkers in ageing-related diseases Scientific Reports |
| title | Optimized RT-qPCR and a novel normalization method for validating circulating miRNA biomarkers in ageing-related diseases |
| title_full | Optimized RT-qPCR and a novel normalization method for validating circulating miRNA biomarkers in ageing-related diseases |
| title_fullStr | Optimized RT-qPCR and a novel normalization method for validating circulating miRNA biomarkers in ageing-related diseases |
| title_full_unstemmed | Optimized RT-qPCR and a novel normalization method for validating circulating miRNA biomarkers in ageing-related diseases |
| title_short | Optimized RT-qPCR and a novel normalization method for validating circulating miRNA biomarkers in ageing-related diseases |
| title_sort | optimized rt qpcr and a novel normalization method for validating circulating mirna biomarkers in ageing related diseases |
| url | https://doi.org/10.1038/s41598-023-47971-3 |
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