Cancer-related mRNA expression analysis using a novel flow cytometry-based assay
| Autor: | Jan Philippé, Barbara De Moerloose, Barbara Denys, Barbara Depreter, Magali Meul, Karl Vandepoele, Tim Lammens |
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| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
Messenger RNA Histology medicine.diagnostic_test Cell RNA Cell Biology Biology medicine.disease Molecular biology Pathology and Forensic Medicine Flow cytometry 03 medical and health sciences 030104 developmental biology medicine.anatomical_structure Cell culture Neuroblastoma Gene expression medicine Gene |
| Zdroj: | Cytometry Part B: Clinical Cytometry. 94:565-575 |
| ISSN: | 1552-4949 |
| DOI: | 10.1002/cyto.b.21593 |
| Popis: | Background. Cancer-related gene expression data mostly originate from unfractionated bulk samples, leading to ‘expression averaging’ of heterogeneous populations. Multicolor flow cytometry (FCM) may distinguish heterogeneous populations based on the phenotypic characterization of single-cells, but is not applicable for RNA targets. Here, we evaluated the PrimeFlow™ RNA assay, a novel FCM-based assay designed to measure gene expressions, in two cancer entities with high and low RNA target levels. Methods. Neuroblastoma (NB) cell lines were studied for MYCN gene expression by PrimeFlow™ and compared with the gold standard, RT-qPCR. Dilution series of NB cells (0.10-11%) were prepared to evaluate performance in small cell populations. Diagnostic material of de novo acute myeloid leukaemia (AML) patients was used to measure Wilms' tumor 1 (WT1) expression in bulk leukemic cells and rare subsets, e.g. leukemic stem cells (LSCs). FCM analysis was performed on a FACSCanto II (BD Biosciences) using Infinicyt™ (Cytognos®) for data analysis. mRNA expression was reported by normalised mean fluorescence intensity (MFI) values and staining indices. Results. MYCN mRNA quantified by PrimeFlow™ significantly correlated with RT-qPCR and remained detectable in small (0.1%) populations. Using PrimeFlowTM, WT1 levels were shown to be significantly higher in AML patient samples with WT1 overexpression, previously defined by RT-qPCR. Moreover, WT1 overexpression was distinguishable between heterogeneous cell populations and remained measurable in rare LSCs. Conclusion. PrimeFlow™ is a sensitive technique to investigate mRNA expressions, with high concordance to RT-qPCR. High (MYCN) and subtle (WT1) overexpressed mRNA targets can be quantified in heterogeneous and rare subpopulations e.g. LSCs. This article is protected by copyright. All rights reserved. |
| Databáze: | OpenAIRE |
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