Assessment of NanoString technology as a tool for profiling circulating miRNA in maternal blood during pregnancy

Circulating maternal miRNA is a promising source of biomarkers for antenatal diagnostics. The hybridisation-based NanoString nCounter is a popular global screening tool due to its simplicity and ease-of-use but there is a lack of standardisation in analysis methods. In this study we used a rodent model to examine the effect of user-defined variables within the data processing software nSolver upon reported changes in maternal blood miRNA during pregnancy. These included the choice of normalisation and background correction methods as well as the background threshold applied. Our results suggest that these variables greatly influence the output of the nSolver software. Changes in a total of 31 miRNAs were detected, but many of these were only picked up by a single combination of analysis variables and no reported change was common to all. RT-qPCR validated changes in 4 miRNAs with known roles in pregnancy (miR-183, miR-196c, miR-431, miR-450a) from a panel of 14 candidates in which changes were supported by multiple analysis workflows. No single nSolver analysis workflow had successfully identified all four validated changes. We then reverse-engineered the nSolver analysis to further investigate the effect of each step in the analysis upon the count data. Our results highlight the need for standardised nCounter data analysis methods and detailed reporting of these methods in papers. We suggest that robust data can only be obtained by examining multiple analysis workflows. Experimental design was 6x6. A control non-pregnant group of 6 rats was compared to a pregnant group of 6 (gestational age E14.5). Maternal blood was obtained by post-mortem cardiac puncture. Total RNA prepared from 1.5ml platelet-free plasma. Osa-miR-414 exogenous spike-in added to samples. Assessment of NanoString technology as a tool for profiling circulating miRNA in maternal blood during pregnancy. Adamova P, Powell AK, Dykes IM. Extracell Vesicles Circ Nucleic Acids. 2024;5:571-96.