Selection of stable reference genes for qPCR-based analysis of circulating cell-free DNA levels in peripheral blood of neuroblastoma patients

As neuroblastoma is a highly heterogeneous pediatric solid tumor, it is essential to select appropriate reference genes to compare the concentration of circulating free DNA (cfDNA) between patients with neuroblastoma and healthy individuals. cfDNA was extracted from peripheral blood and quantified using quantitative Polymerase Chain Reaction (qPCR). The stability of candidate reference genes (RGs) was analyzed using the ΔCt method, geNorm, NormFinder, and BestKeeper. The results from these four algorithms were integrated using the RefFinder tool to generate a comprehensive stability ranking and assess the stability of these candidate RGs across different sample groups. Alpha hemoglobin stabilizing protein (<i>AHSP)</i> and Hypoxanthine-Guanine Phosphoribosyltransferase 1 (<i>HPRT1)</i> exhibited the highest stability, whereas Beta-2-microglobulin(<i>B2M)</i> and HBS1-like protein(<i>HBS1L)</i> demonstrated the lowest stability. qPCR of cfDNA from patients with neuroblastoma revealed differences in the stability of various reference genes. Prioritizing reference genes with better stability may facilitate more accurate detection of intergroup differences in the samples.