Getting at the gene expression of a single cell has long been a goal of researchers hoping to understand how cells interact. "At first, I thought that cells in a tissue would equally express genes and single-cell analysis would be very useful for tissue engineering, including stem cell technology," says Hideki Kambara from the Hitachi Central Research Laboratory in Tokyo.
Kambara and his colleagues have developed a way to quantitatively analyze gene expression in a single cell, as they published in the June issue of Nature Methods. "The gene expression data averaged over ensembles of cells in various stages or timing may mask important matters. Someone said that a gene expressed at a level of 0.5 copies per cell on average sometimes plays an important role in a living system. This might mean that a gene species expressed at a high level in a small number of cells or in some short period plays an important role in a living system, which can be clarified only by single-cell analysis," Kambara says.
To peer into expression in single cells, Kambara and his colleagues attached cDNA libraries to dyna-beads coated with a polymer. For the first couple of PCR rounds, the fragments are the template, but then the beads sink to the bottom. To allow for the beads to be reused, the qPCR reaction progresses at a lower-than-usual temperature — the polymer on the bead is heat-sensitive. They then used their beads to analyze the gene expression levels of four housekeeping genes of cDNA from 14 different single cells as well as from pooled cDNA, finding that the relative expression of the genes was similar but that the absolute amount varied among the individual cells.
Kambara and his team are working to refine this technique — now the beads may be used 20 times and they are hoping to get that up to 100 times — and on a tool to analyze mRNA from thousands of single cells.