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Identification of stably expressed housekeeping miRNAs in endothelial cells and macrophages in an inflammatory setting
Reliable quantification of miRNA expression by qRT-PCR crucially depends on validated housekeepers for data normalization. Here we present thoroughly tested miRNAs eligible as references in immunological studies utilizing endothelial cells and macrophages, respectively. Endothelial cells (cell line:...
Autores principales: | , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Nature Publishing Group UK
2019
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6726651/ https://www.ncbi.nlm.nih.gov/pubmed/31484960 http://dx.doi.org/10.1038/s41598-019-49241-7 |
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author | Link, Fabian Krohn, Knut Schumann, Julia |
author_facet | Link, Fabian Krohn, Knut Schumann, Julia |
author_sort | Link, Fabian |
collection | PubMed |
description | Reliable quantification of miRNA expression by qRT-PCR crucially depends on validated housekeepers for data normalization. Here we present thoroughly tested miRNAs eligible as references in immunological studies utilizing endothelial cells and macrophages, respectively. Endothelial cells (cell line: TIME) and macrophages (cell line: RAW264.7) were treated with various pro- and anti-inflammatory mediators (cytokines, LPS, unsaturated fatty acids) given as either single substances or in combination. Isolated RNA was screened for stably expressed miRNAs by next generation sequencing. Housekeeper candidates were thereafter validated by means of two independent quantification techniques: qRT-PCR for relative quantification and ddPCR for absolute quantification. Both methods consistently confirmed the suitability of let-7g-5p, let-7i-5p, miR-127-3p and miR-151a-5p in cytokine/fatty acid-treated TIME and miR-16-5p, miR-27b-3p, miR-103a-3p and miR-423-3p in LPS/fatty acid-treated RAW264.7, respectively as housekeeping miRNAs. With respect to abundancy and over all expression stability the miRNAs miR-151a-5p (cell line: TIME) as well as miR-27b-3p and miR-103a-3p (cell line: RAW264.7) can be particularly recommended for normalization of qRT-PCR data. |
format | Online Article Text |
id | pubmed-6726651 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2019 |
publisher | Nature Publishing Group UK |
record_format | MEDLINE/PubMed |
spelling | pubmed-67266512019-09-18 Identification of stably expressed housekeeping miRNAs in endothelial cells and macrophages in an inflammatory setting Link, Fabian Krohn, Knut Schumann, Julia Sci Rep Article Reliable quantification of miRNA expression by qRT-PCR crucially depends on validated housekeepers for data normalization. Here we present thoroughly tested miRNAs eligible as references in immunological studies utilizing endothelial cells and macrophages, respectively. Endothelial cells (cell line: TIME) and macrophages (cell line: RAW264.7) were treated with various pro- and anti-inflammatory mediators (cytokines, LPS, unsaturated fatty acids) given as either single substances or in combination. Isolated RNA was screened for stably expressed miRNAs by next generation sequencing. Housekeeper candidates were thereafter validated by means of two independent quantification techniques: qRT-PCR for relative quantification and ddPCR for absolute quantification. Both methods consistently confirmed the suitability of let-7g-5p, let-7i-5p, miR-127-3p and miR-151a-5p in cytokine/fatty acid-treated TIME and miR-16-5p, miR-27b-3p, miR-103a-3p and miR-423-3p in LPS/fatty acid-treated RAW264.7, respectively as housekeeping miRNAs. With respect to abundancy and over all expression stability the miRNAs miR-151a-5p (cell line: TIME) as well as miR-27b-3p and miR-103a-3p (cell line: RAW264.7) can be particularly recommended for normalization of qRT-PCR data. Nature Publishing Group UK 2019-09-04 /pmc/articles/PMC6726651/ /pubmed/31484960 http://dx.doi.org/10.1038/s41598-019-49241-7 Text en © The Author(s) 2019 Open Access This 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 license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license 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 license, visit http://creativecommons.org/licenses/by/4.0/. |
spellingShingle | Article Link, Fabian Krohn, Knut Schumann, Julia Identification of stably expressed housekeeping miRNAs in endothelial cells and macrophages in an inflammatory setting |
title | Identification of stably expressed housekeeping miRNAs in endothelial cells and macrophages in an inflammatory setting |
title_full | Identification of stably expressed housekeeping miRNAs in endothelial cells and macrophages in an inflammatory setting |
title_fullStr | Identification of stably expressed housekeeping miRNAs in endothelial cells and macrophages in an inflammatory setting |
title_full_unstemmed | Identification of stably expressed housekeeping miRNAs in endothelial cells and macrophages in an inflammatory setting |
title_short | Identification of stably expressed housekeeping miRNAs in endothelial cells and macrophages in an inflammatory setting |
title_sort | identification of stably expressed housekeeping mirnas in endothelial cells and macrophages in an inflammatory setting |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6726651/ https://www.ncbi.nlm.nih.gov/pubmed/31484960 http://dx.doi.org/10.1038/s41598-019-49241-7 |
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