A new high-throughput method allows analysis of gene expression in individual cells with high sensitivity, less expensively and more easily than current single-cell sequencing methods.

Not all cells in a tumor are created equal, and cancer researchers increasingly want to study individual cells as a way of understanding heterogeneity at the DNA and RNA level. A new method promises to aid their quest by enabling rapid, massively parallel analysis of gene expression in single cells. The technique accommodates thousands of cells and hundreds of genes per cell at one time with high sensitivity, and does it less expensively and more easily than current single-cell sequencing methods (Science 2015; 347:1258367).

The new method, developed by Stephen P.A. Fodor, PhD, and colleagues at Cellular Research in Palo Alto, CA, relies on standard dilution plating to isolate single cells; each cell is then paired with a single magnetic bead, which is decorated with barcoded mRNA capture probes. Upon cell lysis, the bead captures the cell's mRNA; the beads are then pooled for subsequent reverse transcription, amplification, and sequencing. Because each complementary DNA molecule becomes uniquely tagged, thousands of cells' worth of RNA can be analyzed in parallel, and the resulting sequences can be traced back to their parent cell.

The use of high-density microwell plates makes the method easier to scale up than current technologies involving isolation of single cells on fluidic chips or cell sorting, which are limited to tens or hundreds of cells.

“Single-cell transcriptomics can teach us a great deal about the heterogeneity of tumor populations. However, the throughput in terms of numbers of cells that can be analyzed has lagged,” says Bradley E. Bernstein, MD, PhD, of Massachusetts General Hospital and Harvard Medical School, both in Boston, MA. “This is a problem for identifying relatively rare subsets of cells within a tumor, such as those that may drive tumor propagation or relapse. I find this approach by Fodor and colleagues to be quite clever and exciting for its potential to address this challenge and enable characterization of much larger numbers of cells.”

In their paper, Fodor and lead author H. Christina Fan, PhD, demonstrate the use of the method, CytoSeq, to analyze about 15,000 human blood cells in 12 different experiments. In one experiment, quantifying expression of 93 genes in more than 4,500 immune T cells enabled the team to identify rare antigen-specific cells that occurred at a frequency of 1 in 1,000 cells.

Cellular Research aims to market a CytoSeq system with a capacity of 5 to 10,000 cells per run in 2016, but there is no reason that the technology could not be scaled to the 100,000-cell level, Fodor says. For higher cell numbers and for whole-genome coverage, sequencing capacity would become a limiting factor.

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