Experimental approaches commonly used for single-cell gene expression profiling

Single-cell gene expression profiling was pioneered more than 20 years ago using exponential amplification of cDNAs by PCR and linear amplification by in vitro transcription. These protocols, as well as the subsequent availability of high-density microarrays, led to the application of these technologies to study cellular complexity. New single-cell capture approaches and nucleic acid amplification protocols, coupled with next-generation sequencing (NGS), have permitted the parallel sequencing of a large number of cDNA molecules and scaled down the amount of starting material required for whole transcriptome analysis.

Researchers from Northwestern University review several methods that have been developed to isolate and capture cells for single-cell analysis, including manual or automated micropipetting, cytoplasmic aspiration, laser capture microdissection, FACS, and microfluidic and microdroplet devices. Following library generation, a limited number of genes can be assayed with multiplex qRT-PCR or the transcriptome of a cell can be characterized by RNA-seq. Several bioinformatic approaches allow clustering of cells and assigning identities on the basis of their gene expression profiles.