Cargando…
Reliable reference genes for expression analysis of proliferating and adipogenically differentiating human adipose stromal cells
BACKGROUND: The proliferation and adipogenic differentiation of adipose stromal cells (ASCs) are complex processes comprising major phenotypical alterations driven by up- and downregulation of hundreds of genes. Quantitative RT-PCR can be employed to measure relative changes in the expression of a g...
Autores principales: | , , , , , |
---|---|
Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
BioMed Central
2019
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6377720/ https://www.ncbi.nlm.nih.gov/pubmed/30815013 http://dx.doi.org/10.1186/s11658-019-0140-6 |
_version_ | 1783395799796809728 |
---|---|
author | Krautgasser, Claudia Mandl, Markus Hatzmann, Florian M. Waldegger, Petra Mattesich, Monika Zwerschke, Werner |
author_facet | Krautgasser, Claudia Mandl, Markus Hatzmann, Florian M. Waldegger, Petra Mattesich, Monika Zwerschke, Werner |
author_sort | Krautgasser, Claudia |
collection | PubMed |
description | BACKGROUND: The proliferation and adipogenic differentiation of adipose stromal cells (ASCs) are complex processes comprising major phenotypical alterations driven by up- and downregulation of hundreds of genes. Quantitative RT-PCR can be employed to measure relative changes in the expression of a gene of interest. This approach requires constitutively expressed reference genes for normalization to counteract inter-sample variations due to differences in RNA quality and quantity. Thus, a careful validation of quantitative RT-PCR reference genes is needed to accurately measure fluctuations in the expression of genes. Here, we evaluated candidate reference genes applicable for quantitative RT-PCR analysis of gene expression during proliferation and adipogenesis of human ASCs with the immunophenotype DLK1(+)/CD34(+)/CD90(+)/CD105(+)/CD45(−)/CD31(−). METHODS: We evaluated the applicability of 10 candidate reference genes (GAPDH, TBP, RPS18, EF1A, TFRC, GUSB, PSMD5, CCNA2, LMNA and MRPL19) using NormFinder, geNorm and BestKeeper software. RESULTS: The results indicate that EF1A and MRPL19 are the most reliable reference genes for quantitative RT-PCR analysis of proliferating ASCs. PSMD5 serves as the most reliable endogenous control in adipogenesis. CCNA2 and LMNA were among the least consistent genes. CONCLUSIONS: Applying these findings for future gene expression analyses will help elucidate ASC biology. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1186/s11658-019-0140-6) contains supplementary material, which is available to authorized users. |
format | Online Article Text |
id | pubmed-6377720 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2019 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-63777202019-02-27 Reliable reference genes for expression analysis of proliferating and adipogenically differentiating human adipose stromal cells Krautgasser, Claudia Mandl, Markus Hatzmann, Florian M. Waldegger, Petra Mattesich, Monika Zwerschke, Werner Cell Mol Biol Lett Short Report BACKGROUND: The proliferation and adipogenic differentiation of adipose stromal cells (ASCs) are complex processes comprising major phenotypical alterations driven by up- and downregulation of hundreds of genes. Quantitative RT-PCR can be employed to measure relative changes in the expression of a gene of interest. This approach requires constitutively expressed reference genes for normalization to counteract inter-sample variations due to differences in RNA quality and quantity. Thus, a careful validation of quantitative RT-PCR reference genes is needed to accurately measure fluctuations in the expression of genes. Here, we evaluated candidate reference genes applicable for quantitative RT-PCR analysis of gene expression during proliferation and adipogenesis of human ASCs with the immunophenotype DLK1(+)/CD34(+)/CD90(+)/CD105(+)/CD45(−)/CD31(−). METHODS: We evaluated the applicability of 10 candidate reference genes (GAPDH, TBP, RPS18, EF1A, TFRC, GUSB, PSMD5, CCNA2, LMNA and MRPL19) using NormFinder, geNorm and BestKeeper software. RESULTS: The results indicate that EF1A and MRPL19 are the most reliable reference genes for quantitative RT-PCR analysis of proliferating ASCs. PSMD5 serves as the most reliable endogenous control in adipogenesis. CCNA2 and LMNA were among the least consistent genes. CONCLUSIONS: Applying these findings for future gene expression analyses will help elucidate ASC biology. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1186/s11658-019-0140-6) contains supplementary material, which is available to authorized users. BioMed Central 2019-02-15 /pmc/articles/PMC6377720/ /pubmed/30815013 http://dx.doi.org/10.1186/s11658-019-0140-6 Text en © The Author(s) 2019 Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided 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 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. |
spellingShingle | Short Report Krautgasser, Claudia Mandl, Markus Hatzmann, Florian M. Waldegger, Petra Mattesich, Monika Zwerschke, Werner Reliable reference genes for expression analysis of proliferating and adipogenically differentiating human adipose stromal cells |
title | Reliable reference genes for expression analysis of proliferating and adipogenically differentiating human adipose stromal cells |
title_full | Reliable reference genes for expression analysis of proliferating and adipogenically differentiating human adipose stromal cells |
title_fullStr | Reliable reference genes for expression analysis of proliferating and adipogenically differentiating human adipose stromal cells |
title_full_unstemmed | Reliable reference genes for expression analysis of proliferating and adipogenically differentiating human adipose stromal cells |
title_short | Reliable reference genes for expression analysis of proliferating and adipogenically differentiating human adipose stromal cells |
title_sort | reliable reference genes for expression analysis of proliferating and adipogenically differentiating human adipose stromal cells |
topic | Short Report |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6377720/ https://www.ncbi.nlm.nih.gov/pubmed/30815013 http://dx.doi.org/10.1186/s11658-019-0140-6 |
work_keys_str_mv | AT krautgasserclaudia reliablereferencegenesforexpressionanalysisofproliferatingandadipogenicallydifferentiatinghumanadiposestromalcells AT mandlmarkus reliablereferencegenesforexpressionanalysisofproliferatingandadipogenicallydifferentiatinghumanadiposestromalcells AT hatzmannflorianm reliablereferencegenesforexpressionanalysisofproliferatingandadipogenicallydifferentiatinghumanadiposestromalcells AT waldeggerpetra reliablereferencegenesforexpressionanalysisofproliferatingandadipogenicallydifferentiatinghumanadiposestromalcells AT mattesichmonika reliablereferencegenesforexpressionanalysisofproliferatingandadipogenicallydifferentiatinghumanadiposestromalcells AT zwerschkewerner reliablereferencegenesforexpressionanalysisofproliferatingandadipogenicallydifferentiatinghumanadiposestromalcells |