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Intercepted messages reveal cells' inner workings

23.08.2010
A pair of molecular biology techniques enables detailed characterization of the gene expression of small numbers of cells

A cell’s RNA content provides a complete snapshot of its gene expression activity so can yield a bonanza of information not only about how that cell functions, but also the disruptions that result from disease or environmental changes.

The cap-analysis of gene expression (CAGE) technique developed by Piero Carninci and his colleagues at the RIKEN Omics Science Center in Yokohama has provided an invaluable tool for such profiling, enabling researchers to compile libraries of partial sequences from a large percentage of cellular RNAs1. However, CAGE requires large quantities of genetic material, limiting its usefulness for more focused cellular analyses. “We have been working with neurons, but there are so many types,” says Carninci. “If we isolate specific populations of fluorescently labeled transgenic neurons, we may obtain no more than several thousand cells.”

Working with an international team of collaborators, Carninci’s group has now developed two CAGE variants that bring such analyses within reach2. The first, nanoCAGE, can be applied to as little as ten nanograms of RNA—5,000-fold less than is needed for standard CAGE. Using nanoCAGE, the investigators could even selectively characterize RNAs from the nucleus, nucleolus and other cellular compartments.

Thanks to cellular splicing mechanisms, a single gene can yield multiple, functionally diverse gene products. However, CAGE and nanoCAGE only characterize the beginning of each RNA molecule, making it hard to identify splice variants. The second technique, CAGEscan, has therefore been adapted to yield sequence data from both ends, and initial demonstrations of this method on cultured liver cells enabled a detailed analysis of architecture and revealed a startling diversity of novel RNA molecules. Many of these arise from within non-protein-coding segments of known genes, and potentially exert yet-unknown regulatory functions.

These two techniques should enable a diverse array of genetics and cell biology studies that were not possible with traditional CAGE. “Analyzing isolated, homogeneous neuron populations from mice and rats is a high priority for us,” says Carninci. “These technologies could also be used on biopsies or samples, where the amount of RNA is generally limited or to look for diagnostic markers in the blood.”

Carninci and his colleagues are also thinking smaller, and attempting to focus their method all the way down to the single-cell level. “Scaling down the technology to a single cell will help solve the issue of how many cell types we have in the body,” he says, “and particularly in the brain, where this issue is especially debated.”

The corresponding author for this highlight is based at the Functional Genomic Technology Team, RIKEN Omics Science Center

1. Shiraki, T., Kondo, S., Katayama, S., Waki, K., Kasukawa, T., Kawaji, H., Kodzius, R., Watahiki, A., Nakamura, M., Arakawa, T. et al. Cap analysis gene expression for high-throughput analysis of transcriptional starting point and identification of promoter usage. Proceedings of the National Academy of Sciences USA 100, 15776–15781 (2003).

2. Plessy, C., Bertin, N., Takahashi, H., Simone, R., Salimullah, M., Lassmann, T., Vitezic, M., Severin, J., Olivarius, S., Lazarevic, D. et al. Linking promoters to functional transcripts in small samples with nanoCAGE and CAGEscan. Nature Methods 7, 528–534 (2010).

gro-pr | Research asia research news
Further information:
http://www.rikenresearch.riken.jp/eng/research/6374
http://www.researchsea.com

Further reports about: CAGE CAGEscan Omics RIKEN RNA RNA molecule Science TV cell type environmental change

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