In the last lustrum single-cell techniques such as single-cell quantitative PCR, RNA and DNA sequencing, and the state-of-the-art cytometry by time of flight (CyTOF) mass cytometer have allowed a detailed analysis of the sub-composition of different organs from the bone marrow hematopoietic compartment to the brain. These fine-grained analyses have highlighted the great heterogeneity within each cell compartment revealing previously unknown subpopulations of cells. This fast technological evolution has improved our understanding of the biological processes with a particular focus on rare cells of the immune system.
Schematic diagram of different single-cell techniques. First, a single-cell suspension can be obtained from animal tissues, cultured cells and from clinical samples. For genome-wide transcriptome profiling, cells of interest can be FACS-sorted into multiwell (96 or 384) plates, and library preparation can be performed manually or using a liquid-handling robot (on the left). Cell capturing and library preparation can alternatively be performed using microfluidic devices (middle panel). Single-cell proteomics by mass cytometry can be performed with the cytometry by time of flight (on the right). First, cells are stained with antibodies coupled to isotopically purified mass tags and then analyzed through a mass spectrometer. The individual ions are counted and then resolved into a flow cytometry file format. With this technique, about 40 simultaneous antigens can be quantified in individual cells at a rate of about 500-1000 cells per s. FACS, fluorescence-activated cell sorting.
In particular, the ability of sequencing non polyadenylated RNA and both microRNA and small RNA, the integration of the transcriptomic profile with cell position in three dimensions, the standardization of both the experimental procedure and the computational analysis of the single-cell profiling will be the next crucial steps for enabling a deeper analysis of both the steady-state and the disease mechanisms. (read more…)