Figure 5. Emerging technologies for single-cell or low-quantity-cell gene expression profiling.

a | Single-molecule DNA and RNA sequencing technologies could be modified for single-cell applications. Cells can be delivered to flow cells using fluidics systems, followed by cell lysis and capture of mRNA species on the poly(dT)-coated sequencing surfaces by hybridization. Standard sequencing runs could take place on channels with a 127.5 mm² surface area, requiring 2,750 images to be taken per cycle to image the entire channel area. The surface area needed to accommodate ~350,000 mRNA molecules contained in a single cell is ~0.4 mm²; thus, only eight images per cycle would be needed. Sequence analysis can be done with direct RNA sequencing (DRS)⁷ or on-surface cDNA synthesis followed by single-molecule DNA sequencing²⁶. b | Counter system workflow. Two probes are used for each target site: the capture probe (shown in red) contains a target-specific sequence and a modification that allows the immobilization of the molecules on a surface; the reporter probe contains a different target-specific sequence (shown in blue) and a fluorescent barcode (shown by a green circle) that is unique to each target being examined. After hybridization of the capture and reporter probe mixture to RNA samples in solution, excess probes are removed. The hybridized RNA duplexes are then immobilized on a surface and imaged to identify and count each transcript with the unique fluorescent signals on the capture and reporter probes.