The ability to profile the genome and transcriptome, comprehensively, in single cells, in situ, is crucial to understanding the underlying mechanisms involved in cancer, neurobiology and stem cell biology. However, most of the existing tools can only quantify a small number of different nucleic acids in a sample and gene expression variations and spatial complexity of different RNA species gets masked. Other in situ sequencing based methods may miss transcripts with low copy numbers. Single molecule fluorescence in situ hybridization is a powerful tool for copy number quantification and spatial organization of transcripts in single cells, however, only a few different RNA species in a sample can be simultaneously detected. There is an urgent need for the development of novel, low-cost, multiplexed, single-cell in-situ RNA analysis tools.
Researchers at Arizona State University have developed a low-cost, high-throughput, rapid and high-quality consecutive fluorescence in situ hybridization (C-FISH) method, which will enable the in situ genome and transcriptome-wide analysis in individual cells of intact tissues with single-molecule sensitivity. Furthermore, this method is relatively inexpensive with probe preparation costing about two orders of magnitude lower than conventional systems. This method produces high quality data with single molecule sensitivity and significantly reduced error rates, making it highly versatile and able to overcome the issues with existing systems.
These enhancements to current imaging techniques represent a foundation for advancements in molecular profiling and provide an improvement in molecular diagnoses, targeted therapies, and signal network analysis applications.
Potential Applications
• Molecular diagnosis:
o Cancer diagnosis and prognosis
• Targeted therapies:
o Monitoring the effects of drug treatment among heterogeneous cells
• Basic biological studies
o Systems biology, cell heterogeneity, signaling pathway analysis, etc.
Benefits and Advantages
• Genome or transcriptome-wide analysis
• High-throughput and rapid – reduces the cycling time, except imaging, from weeks to 1.5 hours to analyze thousands of single cells
• Low-cost – the probes are generated by enzymatic amplification of array-derived oligonucleotide libraries, costing about $3/RNA, and $.01/cycle consumables
• High quality data with single-molecule sensitivity – significantly reduced error rates
• Transcripts measured in situ directly to avoid sequence amplification bias – copy number of transcripts determine quantitatively at single molecule sensitivity
For more information about the inventor(s) and their research, please see
Dr. Guo’s laboratory webpage