To do so, he turned to long-time collaborator University of Pennsylvania Professor James Eberwine. Eberwine had pioneered a number of techniques to identify genes active in individual cells.
Sequencing Single Neurons
Bartfai and Eberwine took a unique approach to indentifying gene activity.
Scientists know a gene is "on" in a cell if its messenger RNA (which carries information from genes to sites of protein synthesis) is present. To study gene activity in warm sensitive cells, Eberwine and Bartfai isolated single cells and extracted their RNA. They then made cDNA copies of the messenger RNAs and determined the sequence of the nucleotide bases (adenine, guanine, cytosine, and thymine) in each cDNA molecule.
By matching the DNA sequences obtained to published sequences, the scientists were able to identify the corresponding genes, and thus which genes are turned "on" in the nerve cells.
The technique differs from commonly used methods for studying gene activity. Typically researchers "pool" neurons of one type and examine them as a group, rather than studying single cells. In addition, current techniques generally rely on searching for active genes using microarraysa technique that relies on the preferential binding of sequences in the messenger RNAs /cDNAs to matching DNA sequences "spotted" on the microarray. However, these methods only detect RNAs for which "probes are present on the microarray," in other words, those that are expected. Also, because of the lower sensitivity of this technique than sequencing, only the cDNAs cells produce in relatively large amounts are detected.
"Using single cells, rather than pooling, and sequencing, rather than microarrays, uncovers many more receptors active
|Contact: Mika Ono|
Scripps Research Institute