Authors: Permuth, Jennifer B. | Mesa, Tania | Williams, Sion L. | Cardentey, Yoslayma | Zhang, Dongyu | Pawlak, Erica A. | Li, Jiannong | Cameron, Miles E. | Ali, Karla N. | Jeong, Daniel | Yoder, Sean J. | Chen, Dung-Tsa | Trevino, Jose G. | Merchant, Nipun | Malafa, Mokenge
Article Type:
Research Article
Abstract:
BACKGROUND: Given the growing interest in using microRNAs (miRNAs) as biomarkers of early disease, establishment of robust protocols and platforms for miRNA quantification in biological fluids is critical. OBJECTIVE: The goal of this multi-center pilot study was to evaluate the reproducibility of NanoString nCounter™ technology when analyzing the abundance of miRNAs in plasma and cystic fluid from patients with pancreatic lesions. METHODS: Using sample triplicates analyzed across three study sites, we assessed potential sources of variability (RNA isolation, sample processing/ligation, hybridization, and lot-to-lot variability) that may contribute to suboptimal reproducibility of miRNA abundance when
…using nCounter™, and evaluated expression of positive and negative controls, housekeeping genes, spike-in genes, and miRNAs. RESULTS: Positive controls showed a high correlation across samples from each site (median correlation coefficient, r > 0.9). Most negative control probes had expression levels below background. Housekeeping and spike-in genes each showed a similar distribution of expression and comparable pairwise correlation coefficients of replicate samples across sites. A total of 804 miRNAs showed a similar distribution of pairwise correlation coefficients between replicate samples (p = 0.93). After normalization and selecting miRNAs with expression levels above zero in 80% of samples, 55 miRNAs were identified; heatmap and principal component analysis revealed similar expression patterns and clustering in replicate samples. CONCLUSIONS: Findings from this pilot investigation suggest the nCounter platform can yield reproducible results across study sites. This study underscores the importance of implementing quality control procedures when designing multi-center evaluations of miRNA abundance.
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Keywords: MiRNA quantitation, biomarkers, reproducibility, biospecimens, pancreatic cancer
DOI: 10.3233/CBM-210255
Citation: Cancer Biomarkers,
vol. 33, no. 4, pp. 467-478, 2022