Extended multiplexed fluorescent in situ hybridization by combinatorial encoding of individual transcripts

Abstract

The ability to visualize individual RNA molecules by fluorescent in situ hybridization makes it possible to combinatorially label transcripts. This expands the number of genes whose transcripts can be simultaneously visualized beyond the number of fluorophores used and allows the quantification of gene expression from multiple genes based on transcript counting. We routinely use this to visualize transcripts from six genes simultaneously, but this number is limited only by the number of usable fluorophores and by the resolution of the microscope configuration. Advances in microscope technologies suggest that the number of genes that can be analyzed will increase drastically. Here, we describe the complete process, from the design and preparation of directly labeled oligonucleotide probes to the imaging required to visualize the resulting transcript signals. We also discuss some of the issues that complicate further analysis of the data (e.g., cell segmentation) for which there are presently no simple answers.