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Cancer‐related mRNA expression analysis using a novel flow cytometry‐based assay
Author(s) -
Depreter Barbara,
Philippé Jan,
Meul Magali,
Denys Barbara,
Vandepoele Karl,
De Moerloose Barbara,
Lammens Tim
Publication year - 2018
Publication title -
cytometry part b: clinical cytometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.646
H-Index - 61
eISSN - 1552-4957
pISSN - 1552-4949
DOI - 10.1002/cyto.b.21593
Subject(s) - flow cytometry , myeloid leukemia , messenger rna , microbiology and biotechnology , biology , neuroblastoma , gene expression , leukemia , cancer research , concordance , cancer , gene knockdown , cell culture , gene , immunology , bioinformatics , genetics
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 leukemia (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 normalized 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 PrimeFlow TM , 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. © 2017 International Clinical Cytometry Society