A Microfluidic Platform for High-throughput micro-RNA profiling of single cells
Suman Bose*, Salil Garg, Paige Cole, Jeremy Caplin, Vikash Chauhan, Pillip Sharp, Daniel Anderson
Koch Institute of Integrated Cancer Research, MIT
Several approaches have advanced the ability to sequence messenger-RNA from single cells. MicroRNAs (miRNAs) are small regulatory RNAs that regulate cellular behavior by interacting with thousands of mRNA transcripts. miRNA expressions are less transient compared to mRNA levels and are a robust indicator of cell type and its biological state. miRNA signatures also correlate strongly with disease states. Despite this promise, there are currently no techniques for high-throughput single-cell miRNA sequencing. One challenge is trying to enrich for miRNAs from the rest of nucleic acids, since they only comprise 0.5-3% of all cellular RNAs. Fortunately, biologically active miRNAs are bound to Ago2, so capture of Ago2-miRNA complexes offers a method for isolating miRNAs from rest of the cellular RNAs.
Here we report a microfluidic device that enables enrichment of miRNA from single cells and process them for sequencing. The device works by encapsulating single cells within droplets containing i) magnetic beads coated with anti-Ago2 antibody and ii) a magnetic bead carrying DNA barcodes. The cells are lysed within droplets and released Ago2-miRNAs are captured on the beads. A specially designed microfluidic magnetic tweezer, then isolates the magnetic beads from the larger droplet into a small sub-droplet. The miRNA enriched droplets are heated to release the miRNAs, which are then ligated to the DNA barcodes. Using the device, we demonstrate capture of >40% of miRNAs from single cells and subsequent >15-fold enrichment of miRNA over other cellular RNAs. Finally, we discuss application of the device in single cell miRNA profiling of mouse ES cells.
The ability to profile miRNA in single cells will have implications in many areas of biology and medicine, including identification of new cell types and providing insights into states of cancer stem cells. More generally, this platform technology would enable isolation of any analyte of interest that can be bound by an antibody opening possibility for analysis such as sc-ChIP Seq and sc-proteomics.