Publication date: 5th July 2021
MicroRNAs (miRNA) are a class of short non-coding RNAs (22 nucleotides) that are post-transcriptional regulators. Conventional miRNA detection methods such as northern blotting, next-generation sequencing, and the gold standard, real-time qPCR rely on bulk cell analysis. These methods often also require large cell numbers and do not provide absolute quantification.[1-2] Furthermore, heterogeneity in gene expression and regulation is known to exist within a cell population due to cell-to-cell variation and is often masked when conducting ensemble average measurements. Single cell analysis enables this cellular heterogeneity to be identified and provides a more sensitive method for measuring miRNA levels in scarce, clinical material. It enables the full observation of cellular heterogeneity in cell populations, including the presence of rare cells and ensures that data collected from outlier cells do not bias the data from the cell population.
Previously our group demonstrated the use of the Microfluidic Antigen Capture (MAC) chip to analyse proteins in mammalian and plant cells.[3] We herein report the development of the MAC chip for single cell miRNA quantification. This is achieved by utilizing a sandwich assay that encompasses capture and reporter oligonucleotide probes complementary to the target miRNA sequence of interest, enabling single cell sensitivity and more precise quantification (Figure 1). We initially optimised the chemical surface functionalisation and concentrations of the capture and reporter probes to achieve a low background signal and high capture efficiency. We demonstrate the robustness of this microfluidic technique in capturing and quantifying miR-21 in mammalian cells and primary airway fibroblasts and epithelial cells obtained from chronic obstructive pulmonary disease (COPD) and healthy patients. This novel technique provides large scale screening of hundreds of specific miRNAs at one time and multiple miRNAs can be simultaneously detected, highlighting a novel biological insight towards human microRNA that reveals rich variation at a single cell level.
We would like to thank the Institute of Chemical Biology at Imperial College, and the financial support provided by the Engineering and Physical Sciences Research Council.
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