“When we take a complex tissue and perform expression profiling, we do not necessarily know the cellular origin of the molecules that were quantified,” says assistant professor Peter A. Sims, Ph.D. In cellular populations, biological processes are unlikely to occur in a synchronous fashion, and this opens significant challenges for analyzing those processes and interpreting the data.
One approach to address this shortcoming is to perform analyses at the single-cell level. “We wanted to look at the transcriptomes of individual cells,” explained Dr. Sims. Examining many genes across large numbers of individual cells provides an ideal way to catch a glimpse of biological processes at the single-cell level, and concomitantly capture interindividual heterogeneity in the population.
Ideally, single-cell analysis should be performed by direct detection, to avoid the consequences of amplification bias, and the approach should be time-efficient and affordable. “At this time, we do not have a technology that does not compromise the number of cells analyzed in favor of the number of targets analyzed and vice versa, so it is very difficult to look at a lot of genes and a lot of cells with one tool,” said Dr. Sims.
Investigators in Dr. Sims’ lab rely on two approaches for single-cell analysis, microfluidics and microscopy. Microfluidics uses very small reagent volumes and consequently reduces contamination, whereas microscopy provides additional visual information about the system being examined at a specific time.
By using soft lithography microfluidics, a technology that allows single-cell behavior to be captured under a broad number of conditions, Dr. Sims and colleagues have developed tools for single-cell transcriptome analysis. One of these tools, targeted probe-based expression profiling, offers the possibility of simultaneously exploring several tens of genes within the same cell. “The advantage of this approach is that we are able to probe the transcriptome at the location where the cell is observed,” noted Dr. Sims.
With each cell in its own picoliter-sized chamber, the array can be sealed with a glass surface that is chemically functionalized to allow the capturing of the RNA, which can be reverse transcribed. “We are also developing another tool that uses microarray platforms for large-scale, genome-wide RNA sequencing, where we can generate thousands of cDNA libraries on the microarray well chip,” Dr. Sims said.
Systems biology has made it possible to test hypotheses that merely a few years ago were beyond the reach of experimental approaches. Nonetheless, by working at the juncture of biomedicine, biotechnology, and the clinic, scientists are extending their reach, unveiling mechanistic details, and hastening paradigm shifts. Central to these endeavors, and one of the fundamental teachings that has emerged thus far, is the power of science’s integrative nature.