Improving characterisation of human Multipotent Stromal Cells cultured in 2D and 3D: Design and evaluation of primer sets for accurate gene expression normalisation
| Autor: | Bas Brinkhof, Bo Zhang, Huidong Jia, Zhanfeng Cui, Hui Wang, Hua Ye |
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| Rok vydání: | 2019 |
| Předmět: |
0301 basic medicine
Embryology Molecular biology Gene Expression Biochemistry Umbilical Cord Spectrum Analysis Techniques 0302 clinical medicine Animal Cells Reference genes Gene expression Telomerase Cells Cultured Multidisciplinary Stem Cells Proteases Genomics Flow Cytometry Enzymes RNA isolation Spectrophotometry Trizol 030220 oncology & carcinogenesis Medicine Cytophotometry RNA extraction Cellular Types Pseudogenes Research Article Science Pseudogene Computational biology Biology Real-Time Polymerase Chain Reaction Biomolecular isolation Genome Complexity Marker gene 03 medical and health sciences Extraction techniques Gene Types Genetics Humans Gene Gene Expression Profiling Multipotent Stem Cells Biology and Life Sciences Proteins Computational Biology Mesenchymal Stem Cells Cell Biology Research and analysis methods Gene expression profiling Molecular biology techniques 030104 developmental biology Enzymology Serine Proteases Developmental Biology |
| Zdroj: | PLoS ONE PLoS ONE, Vol 13, Iss 12, p e0209772 (2018) |
| DOI: | 10.1371/journal.pone.0209772 |
| Popis: | Human Multipotent Stromal Cells (MSCs) are a valuable resource for regenerative medicine and are widely studied. They can be isolated from a variety of tissues and differentiate into multiple cell types (multi-potent). Many reports have been published using human MSCs and to be able to compare outcome, or be able to identify differences between MSCs, several cell surface markers have been proposed. Nevertheless, still many differences remain. Gene expression is known to be different between cell stage and origin. Furthermore, cells cultured on a culture dish (2D) show different gene expression profiles as compared to cells grown on scaffolds (3D). Even the RNA extraction method and the selection of genes used for normalisation have a role in gene expression profiling. To be able to compare gene expression data from samples cultured in different dimensions and RNA extracted using a variety of protocols we set out to define a set of reference genes suitable to normalise qPCR data from a very heterogeneous sample set. Hereto, Trizol was used to extract RNA from human MSCs cultured in 3D and 2D to validate newly designed and previously published primer sets. Subsequently, RNA from fresh human MSC samples and samples stored in RLT-buffer, Trizol or RNAlater was extracted using RNeasy and Trizol methods. All samples have been used to rank the candidate reference genes according to their stability after qPCR enabling identification of the most suitable reference gene(s) for normalisation of a heterogeneous sample set. The most stably expressed reference genes indicated superior normalisation of MSC marker gene expression over the least stable reference genes. |
| Databáze: | OpenAIRE |
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