Validating transcripts with probes and imaging technology
In many epithelial tissues, such as the skin and the intestine, there is a clear hierarchical partition into stem cells and diverse differentiated epithelial progenies, each of which displays distinct phenotypic and morphological features.
The precise location of cells within the tissue translates to constant changes in the levels of niche-secreted morphogens, which give rise to position-modulated gene expression programs.
Such measurements allow one to infer the function of genes based on their co-expression patterns.
However, the variability in single cell gene expression in most biological systems and especially in tissues and tumors suggests that bulk transcriptome measurements should be complemented by techniques aimed at characterizing gene expression programs in individual cells Bulk transcriptome measurements inform on the average gene expression in a sample.
High throughput gene expression screens provide a quantitative picture of the average expression signature of biological samples.
However, the analysis of spatial gene expression patterns with single cell resolution requires quantitative hybridization (FISH) techniques that facilitate detection of individual fluorescently labeled m RNA molecules of practically any endogenous gene.
Most solid tumors consist of a mixture of cancer and stromal cells.
Here we present click-amplifying FISH (clamp FISH), a method for fluorescence detection of nucleic acids that achieves high specificity and high-gain (400-fold) signal amplification.These methods, which are based on advances in probe design, imaging technology and image processing, enable the absolute measurement of transcript abundance in individual cells with single-molecule resolution.