Cancer-related mRNA expression analysis using a novel flow cytometry-based assay

Area of Science:

• Single-cell analysis in oncology
• Molecular diagnostics
• Gene expression profiling

Background:

• Cancer gene expression studies often use bulk samples, averaging heterogeneous cell populations.
• Multicolor flow cytometry (FCM) can phenotype single cells but not RNA targets.
• The PrimeFlow™ RNA assay was evaluated for measuring gene expression via FCM.

Purpose of the Study:

• To evaluate the PrimeFlow™ RNA assay for quantifying gene expression in cancer.
• To assess its performance in detecting both high and low RNA target levels.
• To compare its accuracy against established methods like RT-qPCR.

Main Methods:

• Neuroblastoma cell lines were analyzed for MYCN gene expression using PrimeFlow™ and RT-qPCR.
• Dilution series were used to test detection in small cell populations (0.10-11%).
• Acute myeloid leukemia (AML) patient samples were assessed for WT1 expression in bulk and rare subsets (leukemic stem cells).

Main Results:

• PrimeFlow™ accurately quantified MYCN mRNA, correlating well with RT-qPCR and detecting expression in 0.1% cell populations.
• WT1 levels were significantly higher in AML patients with overexpression, as confirmed by RT-qPCR.
• PrimeFlow™ distinguished WT1 overexpression in heterogeneous cells and detected it in rare leukemic stem cells.

Conclusions:

• PrimeFlow™ RNA assay is a sensitive method for mRNA expression analysis with high concordance to RT-qPCR.
• It enables quantification of both highly and subtly overexpressed mRNA targets in heterogeneous and rare cell subpopulations.
• The assay is valuable for analyzing gene expression in complex biological samples, including rare cell populations like LSCs.