Error-correction strategy allows precise measurement of transcriptome in single cells

from Phys.org

rna-seq

Howard Hughes Medical Institute (HHMI) scientists have devised a method of visualizing RNA molecules inside cells so that the identity, location, and abundance of more than 1,000 different RNA species can be determined at the same time. The developers of the new technology say it should be possible to scale up the approach so that tens of thousands of RNA species can be imaged and identified in a single cell.

The approach, called multiplexed error-robust fluorescence in situ hybridization (MERFISH), is described in an article published online April 9, 2015, in Science Express. The technique uses combinatorial labeling, sequential imaging, and error-robust encoding schemes to identify numerous RNA species. In a specific implementation, the authors used tens of thousands of oligonucleotide probes that bind to cellular RNAs to encode each RNA species with a unique combination of readout sequences, and then used fluorescently labeled

readout probes to detect these sequences during 14 or 16 rounds of hybridization. Unique combinations of readout probes bind to individual RNA molecules, spelling out a 14-bit or 16-bit code that identifies each one.

“We feel that we have worked out such a robust approach that we could feasibly scale up to the entire transcriptome [a cell’s complete set of RNAs],” says Xiaowei Zhuang, an HHMI investigator at Harvard University who led the development of the new technique. “In this paper we report measurements of ~1,000 RNA species, already opening up many exciting applications, but there’s no way that we’re stopping here.”

The core transcription machinery of RNA polymerase copies the information found in DNA genes onto messenger RNA molecules that then govern the production of proteins. The abundance of RNAs is commonly measured to indicate the relative activity of specific genes. Additional information can be gleaned by determining where inside a cell or tissue specific RNA molecules are located, Zhuang says, since the RNA location can influence where the encoded protein will perform its function.

An approach known as single-molecule fluorescence in situ hybridization (smFISH) has been valuable for imaging RNA molecules in their natural setting. smFISH, developed by Albert Einstein College of Medicine biologist Robert Singer, uses fluorescent probes made of DNA or RNA to detect specific sequences inside cells. Scientists can use it to quantify and determine the location of specific RNA molecules. By combining multiple probes for each RNA, the method has been used to simutaneously image up to ~30 different RNA molecules in individual cells.

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