Validating transcripts with probes and imaging technology

Iterative rounds of hybridization and click amplify the fluorescence intensity.

We show that clamp FISH enables the detection of RNA species with low-magnification microscopy and in RNA-based flow cytometry.

Thus two adjacent cells could harbor dramatically different expression programs.

Solid tumors represent a particular case of cell heterogeneity.

High-throughput measurements of gene expression on a genomic scale using microarray technology or high throughput sequencing contributed tremendously to our understanding of how genetic networks coordinately function in normal cells and tissues and how they malfunction in disease.

Alternatively a fluorescence activated cell sorter can be used to specifically isolate cells expressing a small number of defined gene expression markers.

These cells can then be used for either bulk measurements or for single cell measurements that require cell lysis, such as quantitative RT-PCR.

Methods for detecting single nucleic acids in cell and tissues, such as fluorescence in situ hybridization (FISH), are limited by relatively low signal intensity and nonspecific probe binding.

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.

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