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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...

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Autores principales: Grätz, Christian, Bui, Maria L. U., Thaqi, Granit, Kirchner, Benedikt, Loewe, Robert P., Pfaffl, Michael W.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2022
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.
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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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