A new study, looking at methods of interpreting gene expression, has identified a new technique that can show the activity of thousands of genes across 10,000 cells, and at single-molecule resolution. The article was published in the recent issue of Nature Methods, called Image-based transcriptomics in thousands of single human cells at single-molecule resolution.
The FISH Technique
The method has been pioneered by two PhD students, Nico Battich and Thomas Stoeger, under the reigns of Professor Lucas Pelkmans, based at the University of Zurich. The team of biologists collaborated in developing a technique that was capable of measuring the “… amount and spatial organization of single transcript molecules in ten thousand single cells.”
Stoeger explains that, during the activation of various genes, transcript molecules are expressed. It is these transcript molecules that offer a means of interpreting genetic expression.
The method relies, firstly, upon the use of a robotic machine to stain these transcription products. Analysis can then be conducted using fluorescent microscopy to visualize the resultant transcription molecules, which glow brightly under the lens.
Specifically, the group used a technique called fluorescent in situ hybridization (FISH), which enables detection of specific genetic sequences, using a complementary strand that is attached to a detectable probe. The technique adds the probe, upon which amplifier and preamplifier DNA can be added, along with DNA-conjugated dye. Simply put, this “branched DNA” form of the FISH method can enhance the amplification of the signal considerably, thereby improving resolution of the image.
Then, up steps ETH Zurich’s supercomputer, Brutus. Brutus is capable of studying one thousand human genes in approximately ten thousand cells. Pelkmans explains that, for the very first time, his group’s research efforts have provided a technique with which to monitor the spatial organization of the transcript molecules produced by a huge array of genes.
Spatial Organization of Transcript Molecules
The researchers were expecting to see vast differences in the amount of transcript molecules between cells. Surprisingly, however, their investigations found there to be disparity in the spatial arrangement of these molecules, within each cell, and when comparing different cells.
Amazingly, the molecules seemed to form unique patterns. In a recent press release, Battich highlighted the relationship between genes that demonstrate similar function and the pattern of the transcription molecules that they express.
“We realized that genes with a similar function also have a similar variability in the transcript patterns… This similarity exceeds the variability in the amount of transcript molecules, and allows us to predict the function of individual genes.”
The team theorize that these patterns in transcript molecules represent a “countermeasure” directed against variability in the quantity of these transcription molecules.
The study also explains the limitations of existing technologies, which use microarrays or RNA-sequencers, and tend to aggregate information from numerous cells. Although analysis of the transcripts of single cells has been achieved in the past, they do not reveal the subcellular region of these transcript molecules.
Ultimately, it is thought that Pelkmans’ research could have important, practical applications in the fields of research and in understanding cancerous tumors. The technique could prove fundamental to mapping the genetic expression within individual cancer cells.
Although measurement of gene activity is common practice in medical diagnosis for a number of conditions, these techniques solely measure quantity of a transcript, but not where they are within the cell. Now, it seems, scientists have a new tool to observe gene expression on a much more specific basis.
By: James Fenner
University of Zurich Press Release