Courtesy of Ethan Bier

Ventral (top) and ventro-lateral (bottom) views of Drosophila embryos triple-labeled to visualize expression patterns. The top image (early blastoderm) shows snail (blue), single minded (red), and rhomboid (green); the bottom image (early gastrulation) shows short gastrulation (blue), ventral nervous system defective (green), and intermediate nervous system defective (red). (Reprinted from http://superfly.ucsd.edu/~davek/gallery.htm)

Scientists at the University of California, San Diego, recently described a novel combination of new and preexisting technology that permits them to visualize five RNAs simultaneously in a single cell.1 The technique, called multiplex labeling, is a variant of multiplex fluorescent in situ hybridization (M-FISH), and was developed by Dave Kosman, a staff scientist in the laboratories of UCSD biologists Ethan Bier and William McGinnis.

Kosman and coworkers, in collaboration with scientists at Molecular Probes (a division of Invitrogen in Carlsbad, Calif.) used the method to directly detect microRNA in an...


Kosman and colleagues also used fluorescent labeling to create a coding system that would identify nascent RNA transcripts, by taking advantage of the gene transcription sites' ability to be visualized as "nuclear dots" via FISH. Both individual fluorophores and multicolor dye combinations were used to expand the number of genes viewed simultaneously. "By using two of those dyes in combination and then overlaying the colors, you can actually make a third color, which expands the multiplex capability of the system greatly," Cox explains.

This combinatorial approach to fluorescence can theoretically detect expression of as many as 50 different genes in a single embryo, says Bier, who explains that multiplex labeling fills a gap between genome-wide expression analysis and ISH techniques that detect one or two genes at a time. "A big need, it seemed, was to fill this gap," he says, "to be able to look at high precision, like you can with in situ hybridization, but with far more than just one or two genes, and be able to unequivocally tell precisely what genes are on in a single cell at a particular time in an embryo in space and in time."

Geneticist Mike Levine at UC, Berkeley, is using multiplex labeling in an attempt to directly visualize long-range enhancer-promoter interactions in Drosophila embryos. In several cases, RNAs associated with an enhancer on one chromosome appeared to interact with those of a promoter on another chromosome, suggesting that the researchers were viewing the process of transvection. This visualization "is possible only with this method," says Levine, who further notes that his entire laboratory is now adopting Kosman's approach over conventional ISH. "The lab [people are] very set in their ways and reluctant to change methods. But they are all switching," Levine says.

Kosman emphasizes that his method is still a work in progress and that several "inconvenient" aspects of the protocol, including the use of antibodies to detect the probes, still need to be worked out. Currently, at most only five genes can be visualized simultaneously without combinatorial fluorescence; Kosman would like to see six or seven at a time. A detailed description of the technology, including protocols, is available at http://superfly.ucsd.edu/~davek.

- Aileen Constans

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