Single cell RT LAMP mRNA detection by integrated droplet sorting and merging.
Recent advances in transcriptomic analysis at single cell resolution reveal cell to cell heterogeneity in a biological sample with unprecedented resolution. Partitioning single cells in individual micro droplets and harvesting each cell's mRNA molecules for next generation sequencing has proven to be an effective method for profiling transcriptomes from a large number of cells at high throughput. However, the assays to recover the full transcriptomes are time consuming in sample preparation and require expensive reagents and sequencing cost. Many biomedical applications, such as pathogen detection, prefer highly sensitive, reliable and low cost detection of selected genes. Here, we present a droplet based microfluidic platform that permits seamless on chip droplet sorting and merging, which enables completing multi step reaction assays within a short time. By sequentially adding lysis buffers and reactant mixtures to micro droplet reactors, we developed a novel workflow of single cell reverse transcription loop mediated isothermal amplification (scRT LAMP) to quantify specific mRNA expression levels in different cell types within one hour. Including single cell encapsulation, sorting, lysing, reactant addition, and quantitative mRNA detection, the fully on chip workflow provides a rapid, robust, and high throughput experimental approach for a wide variety of biomedical studies.
Lab on a chip