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Obtaining Reliable RT-qPCR Results in Molecular Diagnostics—MIQE Goals and Pitfalls for Transcriptional Biomarker Discovery
In this review, we discuss the development pipeline for transcriptional biomarkers in molecular diagnostics and stress the importance of a reliable gene transcript quantification strategy. Hence, a further focus is put on the MIQE guidelines and how to adapt them for biomarker discovery, from signat...
Autores principales: | , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2022
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8953338/ https://www.ncbi.nlm.nih.gov/pubmed/35330136 http://dx.doi.org/10.3390/life12030386 |
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author | Grätz, Christian Bui, Maria L. U. Thaqi, Granit Kirchner, Benedikt Loewe, Robert P. Pfaffl, Michael W. |
author_facet | Grätz, Christian Bui, Maria L. U. Thaqi, Granit Kirchner, Benedikt Loewe, Robert P. Pfaffl, Michael W. |
author_sort | Grätz, Christian |
collection | PubMed |
description | In this review, we discuss the development pipeline for transcriptional biomarkers in molecular diagnostics and stress the importance of a reliable gene transcript quantification strategy. Hence, a further focus is put on the MIQE guidelines and how to adapt them for biomarker discovery, from signature validation up to routine diagnostic applications. First, the advantages and pitfalls of the holistic RNA sequencing for biomarker development will be described to establish a candidate biomarker signature. Sequentially, the RT-qPCR confirmation process will be discussed to validate the discovered biomarker signature. Examples for the successful application of RT-qPCR as a fast and reproducible quantification method in routinemolecular diagnostics are provided. Based on the MIQE guidelines, the importance of “key steps” in RT-qPCR is accurately described, e.g., reverse transcription, proper reference gene selection and, finally, the application of automated RT-qPCR data analysis software. In conclusion, RT-qPCR proves to be a valuable tool in the establishment of a disease-specific transcriptional biomarker signature and will have a great future in molecular diagnostics or personalized medicine. |
format | Online Article Text |
id | pubmed-8953338 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-89533382022-03-26 Obtaining Reliable RT-qPCR Results in Molecular Diagnostics—MIQE Goals and Pitfalls for Transcriptional Biomarker Discovery Grätz, Christian Bui, Maria L. U. Thaqi, Granit Kirchner, Benedikt Loewe, Robert P. Pfaffl, Michael W. Life (Basel) Review In this review, we discuss the development pipeline for transcriptional biomarkers in molecular diagnostics and stress the importance of a reliable gene transcript quantification strategy. Hence, a further focus is put on the MIQE guidelines and how to adapt them for biomarker discovery, from signature validation up to routine diagnostic applications. First, the advantages and pitfalls of the holistic RNA sequencing for biomarker development will be described to establish a candidate biomarker signature. Sequentially, the RT-qPCR confirmation process will be discussed to validate the discovered biomarker signature. Examples for the successful application of RT-qPCR as a fast and reproducible quantification method in routinemolecular diagnostics are provided. Based on the MIQE guidelines, the importance of “key steps” in RT-qPCR is accurately described, e.g., reverse transcription, proper reference gene selection and, finally, the application of automated RT-qPCR data analysis software. In conclusion, RT-qPCR proves to be a valuable tool in the establishment of a disease-specific transcriptional biomarker signature and will have a great future in molecular diagnostics or personalized medicine. MDPI 2022-03-07 /pmc/articles/PMC8953338/ /pubmed/35330136 http://dx.doi.org/10.3390/life12030386 Text en © 2022 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Review Grätz, Christian Bui, Maria L. U. Thaqi, Granit Kirchner, Benedikt Loewe, Robert P. Pfaffl, Michael W. Obtaining Reliable RT-qPCR Results in Molecular Diagnostics—MIQE Goals and Pitfalls for Transcriptional Biomarker Discovery |
title | Obtaining Reliable RT-qPCR Results in Molecular Diagnostics—MIQE Goals and Pitfalls for Transcriptional Biomarker Discovery |
title_full | Obtaining Reliable RT-qPCR Results in Molecular Diagnostics—MIQE Goals and Pitfalls for Transcriptional Biomarker Discovery |
title_fullStr | Obtaining Reliable RT-qPCR Results in Molecular Diagnostics—MIQE Goals and Pitfalls for Transcriptional Biomarker Discovery |
title_full_unstemmed | Obtaining Reliable RT-qPCR Results in Molecular Diagnostics—MIQE Goals and Pitfalls for Transcriptional Biomarker Discovery |
title_short | Obtaining Reliable RT-qPCR Results in Molecular Diagnostics—MIQE Goals and Pitfalls for Transcriptional Biomarker Discovery |
title_sort | obtaining reliable rt-qpcr results in molecular diagnostics—miqe goals and pitfalls for transcriptional biomarker discovery |
topic | Review |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8953338/ https://www.ncbi.nlm.nih.gov/pubmed/35330136 http://dx.doi.org/10.3390/life12030386 |
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