Evaluating Robustness and Sensitivity of the NanoString Technologies nCounter Platform to Enable Multiplexed Gene Expression Analysis of Clinical Samples

Abstract

Analysis of clinical trial specimens such as formalin-fixed paraffin-embedded (FFPE) tissue for molecular mechanisms of disease progression or drug response is often challenging and limited to a few markers at a time. This has led to the increasing importance of highly multiplexed assays that enable profiling of many biomarkers within a single assay. Methods for gene expression analysis have undergone major advances in biomedical research, but obtaining a robust dataset from low-quality RNA samples, such as those isolated from FFPE tissue, remains a challenge. Here, we provide a detailed evaluation of the NanoString Technologies nCounter platform, which provides a direct digital readout of up to 800 mRNA targets simultaneously. We tested this system by examining a broad set of human clinical tissues for a range of technical variables, including sensitivity and limit of detection to varying RNA quantity and quality, reagent performance over time, variability between instruments, the impact of the number of fields of view sampled, and differences between probe sequence locations and overlapping genes across CodeSets. This study demonstrates that Nanostring offers several key advantages, including sensitivity, reproducibility, technical robustness, and utility for clinical application.

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“…6), because it can measure transcripts of both nuclear and plastid encoded genes; it is suitable for measurements with multiple time points with low variability between biological replicates and good dynamic range (Malkov et al, 2009;Veldman-Jones et al, 2015). In our set of genes for interrogation, we included the mRNA for all proteins that had shown a change in response to Fe deficiency (Fig.…”

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Application of omic technologies in cancer research

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“…35 This approach therefore provides a valuable analytical option that delivers results comparable to matched fresh tissue, which is also suitable for archived and partially degraded tissue from FFPE material. 33 Cascione et al used the nanoString nCounter™ platform for the integrated analysis of gene and miRNA expression of triple negative breast cancer (TBNC), and this resulted in the discovery of miRNA expression signatures that can describe phenotypic subtypes within TNBC. 36 A novel promising method for the targeted study of single cells within a tissue sample is offered by nanoString's Digital Spatial Profiling (DSP) technology.…”