Caitlin Smith has a B.A. in biology from Reed College, a Ph.D. in neuroscience from Yale University, and completed postdoctoral work at the Vollum Institute.
Until recently, most DNA sequencing or gene expression studies have analyzed populations of cells. While valuable, such studies yield data that represent an average of all the cells in the population. “Single anything – whether cells or molecules – behave differently than populations,” says Jim Bowlby, co-founder and chief operating officer of Single Cell Technology. “We should not, and indeed do not, measure the same results as we see in a population of N cells divided by N.” Single-cell analysis can reveal previously overlooked differences between cells or among groups of cells, helping scientists to deepen their understanding of cells’ biological systems.
PCR-based single-cell expression profiling
Today single-cell analysis benefits from the exquisite sensitivity of PCR. Real-time, or quantitative, reverse transcription PCR (qRT-PCR) can detect RNA transcripts from single cells, and vendors are supplying researchers with convenient and efficient kits that smooth the workflow. For example, Life Technologies’ Ambion® Single Cell-to-CT™ Kit is designed for gene expression analysis of 1 to 10 cells, says Paco Cifuentes, director of product applications for Life Technologies. The kit includes the necessary tools for each step of the protocol, including sample preparation, reverse transcription, pre-amplification, and qPCR. In addition, Life Technologies’ next generation sequencing platform Ion Torrent™ can sequence DNA from single cells.
The ability to conduct many experiments simultaneously within the same reaction tube by multiplexing is making genomic technologies more efficient. NanoString Technologies recently released its Single Cell Gene Expression Assays for use with their nCounter Analysis System. These assays can simultaneously profile the expression states of up to 800 genes from a single cell. “Highly multiplexed single-cell expression profiling technologies have the potential to profoundly change the single-cell analysis landscape,” says Chris Grimley, NanoString's Vice President of Marketing, Life Sciences. “These technologies have the ability to accelerate the pace of biomarker discovery and allow for identification and characterization of rare cell types that previously have been extremely difficult to profile.”
The analysis of single-cell expression profiling remains challenging, but NanoString’s goal with their nCounter Single Cell Expression Assays is to help researchers quantify interactions between genes. “The ability to model the gene expression of entire pathways will become an increasingly important component of understanding the underlying biology of individual cells,” says Grimley.
Single cells for antibodies
The production of recombinant antibodies – typically a long and expensive process that takes months – is also benefiting from the application of single-cell analysis. In the lab of Harvard University physics professor David Weitz, visiting scientist John Heyman uses water-in-oil droplet microfluidics to isolate for analysis single cells that secrete antibodies of interest. “We then isolate antibody-encoding DNA from these cells to make recombinant antibodies,” he says. “Single cell analysis eliminates the need to immortalize cells prior to assaying the secreted antibody. This makes this process much faster, and allows the use of difficult-to-immortalize cell types, such as plasma cells or antibody-secreting cells isolated from humans.”
Microfluidics are used to sort droplets containing single cells, according to cells having the properties of interest. “Secreted molecules from single compartmentalized cells quickly reach detectable concentrations due to the small droplet volume, which enables the rapid detection and isolation of cells that produce molecules of interest,” says Heyman. The Weitz lab hopes to extend their methods to generating recombinant monoclonal antibodies, which would provide a rich source of these valuable tools. For example, they plan to screen cells from human patients for anti-cancer or anti-viral activity. “We will also be able to screen plasma cells from immunized animals,” says Heyman. “These cells have been shown to be rich sources of immunogen-specific antibody, but they cannot be immortalized and have not been especially useful for commercial antibody development.”
Single Cell Technology also studies antibodies on a large scale – they screen 15,000 antibody secreting cells at once by placing each antibody in an individual 180 pL well, where they study interaction kinetics of the antibody against different molecules. “We then sequence the cognate light and heavy chain of the mRNA variable domains, and sequence mRNA from the signal transduction pathway of reporter cells deposited in the same wells,” says Bowlby. He believes that next-generation sequencing (NGS) will have a profound effect on single-cell analysis. “Just as PCR was a powerful and specific molecular amplifier, NGS enables the unprecedented look at the population of single-cell mRNA, and a population of cells,” he says. “It is the most amazing molecular picture we could ever imagine. Without NGS we could not select unusual and valuable antibodies.”
Microfluidics chips
Fluidigm is smoothing out the single-cell RNA profiling workflow with their automated microfluidics systems, which can analyze up to 96 single cells simultaneously. Fluidigm’s director of single-cell genomics business, Candia Brown, says that the workflow of their C1™ single-cell auto-prep system, from loading a cell to generating data, takes about 13 hours. Using more cells increases the likelihood of statistical significance, and the confidence that any variability you see is biological rather than artifactual. Fluidigm’s systems are also built to reduce variability and increase consistency within and between chips.
Fluidigm’s core technology is a tiny integrated circuit for biologists. “It’s a multilayered chip that’s smaller than a saltine cracker,” explains High. “It’s made up of channels, gates, and chambers. Chambers are exact in their sizing, and the gates allow us to exactly control the flow of both the DNA and chemical reagents. It’s all automated so that 99.9% of the work gets done inside the chip. The key element is that it’s so repeatable and so controllable. Just like a computer chip, we can change the architecture to do different work.”
Among other applications, Fluidigm’s chips are being used to tackle questions about cell heterogeneity. “Even if you have cell types that have similar immunophenotypes, at the molecular level they are already starting to show differential levels of expression,” Brown says. “They also might be showing early indications of making fate decisions. We’re already seeing breakthroughs in characterizing different populations of circulating tumor cells.”
Cifuentes says that single cell analysis is a research area that is growing exponentially, having begun with more sensitive molecular biology tools. “But from the biology point of view,” he says, “I believe that we have just seen the tip of the iceberg of what single cell biology will offer in the future.”
The image at the top of the page is from Life Technologies.