Microfluidic single cell analysis of microRNA levels in small airway epithelial cells and fibroblasts from COPD patients

Abstract

Introduction: COPD is associated with accelerated ageing of the lung due to oxidative stress. Oxidative stress induces miR-34a and miR-21, which are associated with senescent cell phenotype. Conventional methods of measuring miRNA levels are difficult to quantify precisely and require many cells. Microfluidic assays enable precise quantification miRNAs in single cells (SC) using oligonucleotide probes.

Aim: To develop an assay to quantify SC miRNA and compare with RT-qPCR in COPD cells

Methods: Single cells (50 cells/experiment) were lysed and target miRNA molecules captured, bound to fluorescently tagged probes and detected using fluorescence. RT-qPCR (100,000 cells) was compared with SC assays using primary small airway fibroblasts (SAF) and epithelial cells (SAEC) from COPD patients and non-smokers (NS).

Results: miR-21 and miR-34a were increased in COPD SAF compared with cells from NS (miR-21: 70439±7254 vs 19578± 4529 molecules/cell, p<0.05; miR-34a: 60386±7124 vs 3028±786 molecules/cell, p<0.05 n=8), confirmed with RT-qPCR miR-21 and miR-34a increased by 2-fold and 5-fold (p<0.05 n=5) respectively. Similarly, both miRNAs were increased in COPD SAEC compared with NS (miR-21: 103939±9127 vs 45107±3604, p<0.05; miR-34a: 83116±12603 vs 5510±963 molecules/cell p<0.05 n=4), and with RT-qPCR miR-21 and miR-34a increased by 6-fold and 18-fold (p<0.05 n=4) respectively.

Conclusion: Absolute quantification of miRNA expression can be measured in SC. These data confirm that miR-21 and miR-34a are elevated in COPD SAF and SAEC compared to NS but this can be measured in 50 cells, suggesting SC analysis might be useful for detecting senescent cells and effects of therapy.