Specific and Sensitive Detection of Neuroblastoma mRNA Markers by Multiplex RT-qPCR.
Lieke M J van ZogchelLily Zappeij-KannegieterAhmad JavadiMarjolein LugtigheidNina U GelineauNathalie S M LakDanny A ZwijnenburgJan KosterJanine StutterheimC Ellen van der SchootGodelieve A M TytgatPublished in: Cancers (2021)
mRNA RT-qPCR is shown to be a very sensitive technique to detect minimal residual disease (MRD) in patients with neuroblastoma. Multiple mRNA markers are known to detect heterogeneous neuroblastoma cells in bone marrow (BM) or blood from patients. However, the limited volumes of BM and blood available can hamper the detection of multiple markers. To make optimal use of these samples, we developed a multiplex RT-qPCR for the detection of MRD in neuroblastoma. GUSB and PHOX2B were tested as single markers. The adrenergic markers TH, GAP43, CHRNA3 and DBH and mesenchymal markers POSTN, PRRX1 and FMO3 were tested in multiplex. Using control blood and BM, we established new thresholds for positivity. Comparison of multiplex and singleplex RT-qPCR results from 21 blood and 24 BM samples from neuroblastoma patients demonstrated a comparable sensitivity. With this multiplex RT-qPCR, we are able to test seven different neuroblastoma mRNA markers, which overcomes tumor heterogeneity and improves sensitivity of MRD detection, even in those samples of low RNA quantity. With resources and time being saved, reduction in sample volume and consumables can assist in the introduction of MRD by RT-qPCR into clinical practice.
Keyphrases
- real time pcr
- bone marrow
- end stage renal disease
- sensitive detection
- high throughput
- loop mediated isothermal amplification
- newly diagnosed
- peritoneal dialysis
- ejection fraction
- chronic kidney disease
- induced apoptosis
- prognostic factors
- binding protein
- quantum dots
- label free
- patient reported
- single cell
- cell proliferation
- endoplasmic reticulum stress
- cell cycle arrest