Genome-wide transcriptome analyses are routinely used to monitor tissue-, disease- and cell type-specific gene expression, but it has been technically challenging to generate expression profiles from single cells.
Now, a team of researchers led by scientists from the Ludwig Institute and the Karolinska Institute, Sweden, in collaboration with Illumina have developed a robust mRNA-Seq protocol (Smart-Seq) that is applicable down to single cell levels. Compared with existing methods, Smart-Seq has improved read coverage across transcripts, which enhances detailed analyses of alternative transcript isoforms and identification of single-nucleotide polymorphisms.
The scientists determined the sensitivity and quantitative accuracy of Smart-Seq for single-cell transcriptomics by evaluating it on total RNA dilution series. They found that although gene expression estimates from single cells have increased noise, hundreds of differentially expressed genes could be identified using few cells per cell type. They next applied Smart-Seq to circulating tumor cells from melanomas and identified distinct gene expression patterns, including candidate biomarkers for melanoma circulating tumor cells. This protocol will be useful for addressing fundamental biological problems requiring genome-wide transcriptome profiling in rare cells.
- Ramsköld D, Luo S, Wang YC, Li R, Deng Q, Faridani OR, Daniels GA, Khrebtukova I, Loring JF, Laurent LC, Schroth GP, Sandberg R. (2012) Full-length mRNA-Seq from single-cell levels of RNA and individual circulating tumor cells. Nat Biotechnol [Epub ahead of print]. [abstract]