An amplification-free, innovative, multiplex assay to quantify microRNAs in single cells from COPD patients

<b>Introduction:</b> Chronic obstructive pulmonary disease (COPD) is associated with accelerated ageing of the lung. In COPD, microRNA(miR)-21 is up-regulated in plasma and miR-34a in epithelial cells. Microfluidic technology enables multiplexing of miRNA assays with single cell analysis to absolutely quantify miRNAs in scarce samples. <b>Aim:</b> To develop a multiplex assay to absolutely quantify miR-21 and miR-34a molecules from the same cell <b>Methods:</b> A multiplex chip was microarrayed with miR-21 and miR-34a capture probes. Complimentary reporter probes were tagged with different fluorophores. Small airway fibroblasts (SAF) and epithelial cells (SAEC) (50 cells/experiment) were optically manipulated and lysed. Single miR-21 and miR-34a molecules released from the same cell were imaged using microscopy. <b>Results:</b> MiR-21 expression in COPD was significantly elevated (SAF: 83.5±1.2 x10³ and SAEC: 102±0.9 x10³, p&lt;0.05) compared to non-smokers (NS) (SAF: 27.5±0.3 x10³ and SAEC: 23.5±0.4 x10³) molecules/cell. MiR-34a expression in COPD was significantly increased (SAF: 65.1±0.7 x10³ and SAEC: 79.8±0.4 x10³, p&lt;0.05) compared to NS (SAF: 3.1±0.01 x10³ and SAEC: 7.42±0.1 x10³) molecules/cell. Both miRNAs expression were positively correlated in SAF and SAEC but reduced in COPD SAF (NS: r=0.81, p&lt;0.0001 vs COPD: r= 0.66, p&lt;0.0001, n=5) and SAEC (NS: r=0.86, p&lt;0.0001 vs COPD: r=0.64, p&lt;0.0001, n=5). <b>Conclusion:</b> Absolute expression of miRNAs can be quantified from the same cell using a microfluidic multiplex assay. Expression of miR-21 and miR-34a in a single cell are positively correlated&nbsp;but differ&nbsp;between COPD and NS subjects. Thus, this novel tool may be used to identify miRNAs as COPD biomarkers.