Cargando…
A Strategy for the Selection of RT-qPCR Reference Genes Based on Publicly Available Transcriptomic Datasets
In the next-generation sequencing era, RT-qPCR is still widely employed to quantify levels of nucleic acids of interest due to its popularity, versatility, and limited costs. The measurement of transcriptional levels through RT-qPCR critically depends on reference genes used for normalization. Here,...
Autores principales: | , , , , , , , , , |
---|---|
Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2023
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10135859/ https://www.ncbi.nlm.nih.gov/pubmed/37189697 http://dx.doi.org/10.3390/biomedicines11041079 |
_version_ | 1785032081248616448 |
---|---|
author | Nevone, Alice Lattarulo, Francesca Russo, Monica Panno, Giada Milani, Paolo Basset, Marco Avanzini, Maria Antonietta Merlini, Giampaolo Palladini, Giovanni Nuvolone, Mario |
author_facet | Nevone, Alice Lattarulo, Francesca Russo, Monica Panno, Giada Milani, Paolo Basset, Marco Avanzini, Maria Antonietta Merlini, Giampaolo Palladini, Giovanni Nuvolone, Mario |
author_sort | Nevone, Alice |
collection | PubMed |
description | In the next-generation sequencing era, RT-qPCR is still widely employed to quantify levels of nucleic acids of interest due to its popularity, versatility, and limited costs. The measurement of transcriptional levels through RT-qPCR critically depends on reference genes used for normalization. Here, we devised a strategy to select appropriate reference genes for a specific clinical/experimental setting based on publicly available transcriptomic datasets and a pipeline for RT-qPCR assay design and validation. As a proof-of-principle, we applied this strategy to identify and validate reference genes for transcriptional studies of bone-marrow plasma cells from patients with AL amyloidosis. We performed a systematic review of published literature to compile a list of 163 candidate reference genes for RT-qPCR experiments employing human samples. Next, we interrogated the Gene Expression Omnibus to assess expression levels of these genes in published transcriptomic studies on bone-marrow plasma cells from patients with different plasma cell dyscrasias and identified the most stably expressed genes as candidate normalizing genes. Experimental validation on bone-marrow plasma cells showed the superiority of candidate reference genes identified through this strategy over commonly employed “housekeeping” genes. The strategy presented here may apply to other clinical and experimental settings for which publicly available transcriptomic datasets are available. |
format | Online Article Text |
id | pubmed-10135859 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-101358592023-04-28 A Strategy for the Selection of RT-qPCR Reference Genes Based on Publicly Available Transcriptomic Datasets Nevone, Alice Lattarulo, Francesca Russo, Monica Panno, Giada Milani, Paolo Basset, Marco Avanzini, Maria Antonietta Merlini, Giampaolo Palladini, Giovanni Nuvolone, Mario Biomedicines Article In the next-generation sequencing era, RT-qPCR is still widely employed to quantify levels of nucleic acids of interest due to its popularity, versatility, and limited costs. The measurement of transcriptional levels through RT-qPCR critically depends on reference genes used for normalization. Here, we devised a strategy to select appropriate reference genes for a specific clinical/experimental setting based on publicly available transcriptomic datasets and a pipeline for RT-qPCR assay design and validation. As a proof-of-principle, we applied this strategy to identify and validate reference genes for transcriptional studies of bone-marrow plasma cells from patients with AL amyloidosis. We performed a systematic review of published literature to compile a list of 163 candidate reference genes for RT-qPCR experiments employing human samples. Next, we interrogated the Gene Expression Omnibus to assess expression levels of these genes in published transcriptomic studies on bone-marrow plasma cells from patients with different plasma cell dyscrasias and identified the most stably expressed genes as candidate normalizing genes. Experimental validation on bone-marrow plasma cells showed the superiority of candidate reference genes identified through this strategy over commonly employed “housekeeping” genes. The strategy presented here may apply to other clinical and experimental settings for which publicly available transcriptomic datasets are available. MDPI 2023-04-03 /pmc/articles/PMC10135859/ /pubmed/37189697 http://dx.doi.org/10.3390/biomedicines11041079 Text en © 2023 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 | Article Nevone, Alice Lattarulo, Francesca Russo, Monica Panno, Giada Milani, Paolo Basset, Marco Avanzini, Maria Antonietta Merlini, Giampaolo Palladini, Giovanni Nuvolone, Mario A Strategy for the Selection of RT-qPCR Reference Genes Based on Publicly Available Transcriptomic Datasets |
title | A Strategy for the Selection of RT-qPCR Reference Genes Based on Publicly Available Transcriptomic Datasets |
title_full | A Strategy for the Selection of RT-qPCR Reference Genes Based on Publicly Available Transcriptomic Datasets |
title_fullStr | A Strategy for the Selection of RT-qPCR Reference Genes Based on Publicly Available Transcriptomic Datasets |
title_full_unstemmed | A Strategy for the Selection of RT-qPCR Reference Genes Based on Publicly Available Transcriptomic Datasets |
title_short | A Strategy for the Selection of RT-qPCR Reference Genes Based on Publicly Available Transcriptomic Datasets |
title_sort | strategy for the selection of rt-qpcr reference genes based on publicly available transcriptomic datasets |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10135859/ https://www.ncbi.nlm.nih.gov/pubmed/37189697 http://dx.doi.org/10.3390/biomedicines11041079 |
work_keys_str_mv | AT nevonealice astrategyfortheselectionofrtqpcrreferencegenesbasedonpubliclyavailabletranscriptomicdatasets AT lattarulofrancesca astrategyfortheselectionofrtqpcrreferencegenesbasedonpubliclyavailabletranscriptomicdatasets AT russomonica astrategyfortheselectionofrtqpcrreferencegenesbasedonpubliclyavailabletranscriptomicdatasets AT pannogiada astrategyfortheselectionofrtqpcrreferencegenesbasedonpubliclyavailabletranscriptomicdatasets AT milanipaolo astrategyfortheselectionofrtqpcrreferencegenesbasedonpubliclyavailabletranscriptomicdatasets AT bassetmarco astrategyfortheselectionofrtqpcrreferencegenesbasedonpubliclyavailabletranscriptomicdatasets AT avanzinimariaantonietta astrategyfortheselectionofrtqpcrreferencegenesbasedonpubliclyavailabletranscriptomicdatasets AT merlinigiampaolo astrategyfortheselectionofrtqpcrreferencegenesbasedonpubliclyavailabletranscriptomicdatasets AT palladinigiovanni astrategyfortheselectionofrtqpcrreferencegenesbasedonpubliclyavailabletranscriptomicdatasets AT nuvolonemario astrategyfortheselectionofrtqpcrreferencegenesbasedonpubliclyavailabletranscriptomicdatasets AT nevonealice strategyfortheselectionofrtqpcrreferencegenesbasedonpubliclyavailabletranscriptomicdatasets AT lattarulofrancesca strategyfortheselectionofrtqpcrreferencegenesbasedonpubliclyavailabletranscriptomicdatasets AT russomonica strategyfortheselectionofrtqpcrreferencegenesbasedonpubliclyavailabletranscriptomicdatasets AT pannogiada strategyfortheselectionofrtqpcrreferencegenesbasedonpubliclyavailabletranscriptomicdatasets AT milanipaolo strategyfortheselectionofrtqpcrreferencegenesbasedonpubliclyavailabletranscriptomicdatasets AT bassetmarco strategyfortheselectionofrtqpcrreferencegenesbasedonpubliclyavailabletranscriptomicdatasets AT avanzinimariaantonietta strategyfortheselectionofrtqpcrreferencegenesbasedonpubliclyavailabletranscriptomicdatasets AT merlinigiampaolo strategyfortheselectionofrtqpcrreferencegenesbasedonpubliclyavailabletranscriptomicdatasets AT palladinigiovanni strategyfortheselectionofrtqpcrreferencegenesbasedonpubliclyavailabletranscriptomicdatasets AT nuvolonemario strategyfortheselectionofrtqpcrreferencegenesbasedonpubliclyavailabletranscriptomicdatasets |