Method to visualize RNA molecules in individual cells
February 27, 2014 § Leave a comment
Having a map of where RNA molecules are within single cells will help scientists better understand the dynamics of gene expression, which can differ significantly between cells. The map also can reveal how single-cell dynamics change under different physiological conditions, such as development and growth, or during disease processes, such as in various cancers.
The method, described by a team led by Je Hyuk Lee and George Church at Harvard Medical School, is called fluorescent in situ RNA sequencing, or FISSEQ. Church says that for the method to be successful the investigators knew that they had to be able to sequence individual RNAs as well as hold the material steady and still at their locations.
In the current work, the researchers developed a way to cross-link amplified molecules of cDNA generated from the RNA transcripts into a highly stable matrix inside a cell. They imaged those cross-linked molecules using fluorescence microscopy and were able to tell where active gene expression was happening in the various parts of the cell.
As proof of concept, Lee, Church and colleagues obtained reads of 30 bases from more than 8,000 genes in human primary fibroblasts. They showed that they could track changes in expression in those cells with a simulated wound-healing assay.
The researchers have applied FISSEQ to other cell types, including mouse embryos, mouse brain sections, whole-mount Drosophila embryos and human stem cells. “It seems to work on every situation that we’ve tried,” says Church.
Church says because the FISSEQ seems to work in most sample types, “clinical pathology specimens should be directly adaptable to FISSEQ. We can learn if previously similar-looking cells are actually molecularly significantly different.” He adds that the data could reveal the heterogeneity in tumors, inflammatory processes and growth of blood vessels
At the moment, the researchers are applying FISSEQ to the Brain Research through Advancing Innovative Neurotechnologies, or BRAIN, Initiative that was rolled out by the Obama administration last year. Church says they are collecting “diverse types of data — RNA, cell connections, developmental lineages and time records of the signals going through those connections — all integrated into a single brain sample with related behavioral data.”