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Unbiased RNA-Seq-driven identification and validation of reference genes for quantitative RT-PCR analyses of pooled cancer exosomes

BACKGROUND: Exosomes are extracellular vesicles (EVs) derived from endocytic compartments of eukaryotic cells which contain various biomolecules like mRNAs or miRNAs. Exosomes influence the biologic behaviour and progression of malignancies and are promising candidates as non-invasive diagnostic bio...

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Autores principales: Dai, Yao, Cao, Yumeng, Köhler, Jens, Lu, Aiping, Xu, Shaohua, Wang, Haiyun
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7789813/
https://www.ncbi.nlm.nih.gov/pubmed/33407103
http://dx.doi.org/10.1186/s12864-020-07318-y
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author Dai, Yao
Cao, Yumeng
Köhler, Jens
Lu, Aiping
Xu, Shaohua
Wang, Haiyun
author_facet Dai, Yao
Cao, Yumeng
Köhler, Jens
Lu, Aiping
Xu, Shaohua
Wang, Haiyun
author_sort Dai, Yao
collection PubMed
description BACKGROUND: Exosomes are extracellular vesicles (EVs) derived from endocytic compartments of eukaryotic cells which contain various biomolecules like mRNAs or miRNAs. Exosomes influence the biologic behaviour and progression of malignancies and are promising candidates as non-invasive diagnostic biomarkers or as targets for therapeutic interventions. Usually, quantitative real-time polymerase chain reaction (qRT-PCR) is used to assess gene expression in cancer exosomes, however, the ideal reference genes for normalization yet remain to be identified. RESULTS: In this study, we performed an unbiased analysis of high-throughput mRNA and miRNA-sequencing data from exosomes of patients with various cancer types and identify candidate reference genes and miRNAs in cancer exosomes. The expression stability of these candidate reference genes was evaluated by the coefficient of variation “CV” and the average expression stability value “M”. We subsequently validated these candidate reference genes in exosomes from an independent cohort of ovarian cancer patients and healthy control individuals by qRT-PCR. CONCLUSIONS: Our study identifies OAZ1 and hsa-miR-6835-3p as the most reliable individual reference genes for mRNA and miRNA quantification, respectively. For superior accuracy, we recommend the use of a combination of reference genes - OAZ1/SERF2/MPP1 for mRNA and hsa-miR-6835-3p/hsa-miR-4468-3p for miRNA analyses.
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spelling pubmed-77898132021-01-11 Unbiased RNA-Seq-driven identification and validation of reference genes for quantitative RT-PCR analyses of pooled cancer exosomes Dai, Yao Cao, Yumeng Köhler, Jens Lu, Aiping Xu, Shaohua Wang, Haiyun BMC Genomics Research Article BACKGROUND: Exosomes are extracellular vesicles (EVs) derived from endocytic compartments of eukaryotic cells which contain various biomolecules like mRNAs or miRNAs. Exosomes influence the biologic behaviour and progression of malignancies and are promising candidates as non-invasive diagnostic biomarkers or as targets for therapeutic interventions. Usually, quantitative real-time polymerase chain reaction (qRT-PCR) is used to assess gene expression in cancer exosomes, however, the ideal reference genes for normalization yet remain to be identified. RESULTS: In this study, we performed an unbiased analysis of high-throughput mRNA and miRNA-sequencing data from exosomes of patients with various cancer types and identify candidate reference genes and miRNAs in cancer exosomes. The expression stability of these candidate reference genes was evaluated by the coefficient of variation “CV” and the average expression stability value “M”. We subsequently validated these candidate reference genes in exosomes from an independent cohort of ovarian cancer patients and healthy control individuals by qRT-PCR. CONCLUSIONS: Our study identifies OAZ1 and hsa-miR-6835-3p as the most reliable individual reference genes for mRNA and miRNA quantification, respectively. For superior accuracy, we recommend the use of a combination of reference genes - OAZ1/SERF2/MPP1 for mRNA and hsa-miR-6835-3p/hsa-miR-4468-3p for miRNA analyses. BioMed Central 2021-01-06 /pmc/articles/PMC7789813/ /pubmed/33407103 http://dx.doi.org/10.1186/s12864-020-07318-y Text en © The Author(s) 2020 Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.
spellingShingle Research Article
Dai, Yao
Cao, Yumeng
Köhler, Jens
Lu, Aiping
Xu, Shaohua
Wang, Haiyun
Unbiased RNA-Seq-driven identification and validation of reference genes for quantitative RT-PCR analyses of pooled cancer exosomes
title Unbiased RNA-Seq-driven identification and validation of reference genes for quantitative RT-PCR analyses of pooled cancer exosomes
title_full Unbiased RNA-Seq-driven identification and validation of reference genes for quantitative RT-PCR analyses of pooled cancer exosomes
title_fullStr Unbiased RNA-Seq-driven identification and validation of reference genes for quantitative RT-PCR analyses of pooled cancer exosomes
title_full_unstemmed Unbiased RNA-Seq-driven identification and validation of reference genes for quantitative RT-PCR analyses of pooled cancer exosomes
title_short Unbiased RNA-Seq-driven identification and validation of reference genes for quantitative RT-PCR analyses of pooled cancer exosomes
title_sort unbiased rna-seq-driven identification and validation of reference genes for quantitative rt-pcr analyses of pooled cancer exosomes
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7789813/
https://www.ncbi.nlm.nih.gov/pubmed/33407103
http://dx.doi.org/10.1186/s12864-020-07318-y
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