Systematic evaluation of multiple qPCR platforms, NanoString and miRNA-Seq for microRNA biomarker discovery in human biofluids

Abstract

Aberrant miRNA expression has been associated with many diseases, and extracellular miRNAs that circulate in the bloodstream are remarkably stable. Recently, there has been growing interest in identifying cell-free circulating miRNAs that can serve as non-invasive biomarkers for early detection of disease or selection of treatment options. However, quantifying miRNA levels in biofluids is technically challenging due to their low abundance. Using reference samples, we performed a cross-platform evaluation in which miRNA profiling was performed on four different qPCR platforms (MiRXES, Qiagen, Applied Biosystems, Exiqon), nCounter technology (NanoString), and miRNA-Seq. Overall, our results suggest that using miRNA-Seq for discovery and targeted qPCR for validation is a rational strategy for miRNA biomarker development in clinical samples that involve limited amounts of biofluids.

Figure 1

Overview of microRNA quantitation platforms, RNA samples, and different aspects of platform performance that were assessed in this study. Other qPCR platforms are Qiagen, ABI and Exiqon.

Figure 2

Detection rate, inter-platform overlap and LLOQ. (a) Detection rate of miRNAs as a function of sequencing depth for Brain QC and Ref. Serum miRNA-Seq libraries. (b) LLOQ of Brain QC and Ref. Serum miRNA-Seq libraries as a function of sequencing depth. LLOQ is expressed as the number of mapped reads per miRNA. (c) Coefficient of variation (%CV) observed in expression level for miRNAs detected above the LLOQ. (d) Overlap in miRNAs detected above LLOQ across platforms.

Figure 3

Inter-platform correlation. Pairwise inter-platform correlation in expression levels for miRNAs that were detected above the LLOQ in Ref. Serum. The miRNAs measured by qPCR do not include those in the “expanded” panel (see text). r Spearman’s correlation coefficient, ns not significant (p > 0.05).

Figure 4

Validation of 14 novel miRNAs identified in Ref. Serum. (a) Correlation in expression levels of predicted mature miRNAs measured by qPCR and miRNA-Seq in Ref. Serum. Two miRNAs were not detected above the LOD (data points not shown). r Spearman’s correlation coefficient. (b) Intracellular expression levels of 13 novel miRNAs measured by qPCR across six human cell lines (left y-axis) and read count measured by miRNA-Seq in Ref. Serum (right y-axis). One miRNA was not detected above LOD (data points not shown). (c) Extracellular expression levels of nine novel miRNAs measured by qPCR across six human cell lines (left y-axis) and read count measured by miRNA-Seq in Ref. Serum (right y-axis). Five miRNAs were not detected above LOD (data points not shown). (d) Ratio of extracellular to intracellular expression.