In recent years, heterogeneity in cell populations has brought about a new area of research, focused on exploring single cells. By looking at nucleic acids from a mixed sample, you can only see information based on cell averages, even though there may be low-frequency differences in cell subpopulations. By analyzing on a single-cell level, we can discern the impact of genomic or transcriptomic differences during disease evolution or cell differentiation in normal development.
Single-cell analysis is a great tool for researchers, but getting individual cells without using harsh methods that can lead to cell death or methods that lack precision and reproducibility can be a problem.
New, novel methodology
QIAGEN has recently released a novel technology that combines elements to maximize efficiency, cell viability and affordability. This new system is called the QIAscout, which uses microraft technology to singularize cells in an environment similar to a standard cell culture dish. Basically, the protocol begins by adding a cell suspension to the QIAscout Array. Cells distribute equally across the array and actively adhere to the raft with many rafts containing one single cell. Cells of interest are identified based on certain criterion, such as cell morphology or fluorescent labeling. Then, using a fine needle mounted on an inverted microscope, the raft containing the cell of interest is “released” from the array with the push of a button. The microraft with the attached cell – now freed from the array – is magnetic and can easily picked out using a magnetic wand and dropped into a tube or 96-well plate for further analysis or cultivation. You can watch a quick, 3-minute animation on the process here.
We wanted to compare the ability to pick a single cell using the new QIAscout process with two other common single-cell isolation methods: serial dilution and pipetting. Serial dilution is a commonly-used and inexpensive method for isolating a single cell, which involves creating a serial dilution of a population to the point of isolating only one cell (1). Although this process is simple, cheap and requires only basic pipetting tools and training, researchers can encounter a number of issues that can complicate a single-cell experiment – for example, during transfer to a tube, cells may end up sticking to the pipet. The precision and reproducibility of serial dilution is just not there; the probability of isolating a single cell, rather than multiple cells or even no cell, is low and needs to be tested via seeding (1). Pipetting, or manual cell picking, utilizes a microscope to visualize a sample. The user can then identify the cell of interest and employ a micropipette to aspirate the single cell and transfer to a well or plate (1). The throughput with this method tends to be very limited and requires special expertise to perform (2). The main challenge of this method is to aspirate only one single cell and not multiple cells or even no cell.
Considering these issues, researchers need a single-cell isolation solution that is affordable, user-friendly and accurate.
Two experiments, one conclusion
In order to do this comparison and look at the QIAscout’s performance, we performed two experiments to isolate cells of interest based on two common cell criteria: morphology or fluorescent labeling.
To look at isolating a cell based on fluorescence, we prepared three different cell lines: 1) HT-29; 2) LoVo; and 3) MDA-MB-231 (red fluorescent cells).
All 3 cell lines were added to a mixture in equal ratios and the fluorescent cells were targeted for isolation using the QIAscout, pipetting and serial dilution methods. Once isolated, whole genome amplification was performed on each expected single cell and DNA was analyzed using pyrosequencing to identify the three cell-line specific mutations.
In our second experiment, we also wanted to look at how effective the QIAscout would be if cells were selected based on morphology – rather than fluorescence – since this is another popular means of selecting cells of interest. Similar to our first experiment, we prepared 3 cell lines that were distinguishable by morphological characteristics in a mixture with equal ratios: 1) HT-29 (round cells); LoVo (elongated cells); and SW48 (round cells)
We took this mixture and used our 3 methods, the QIAscout, pipetting and serial dilution, to target the elongated cells (i.e. LoVo cells). Once single cells were isolated, they were amplified using whole genome amplification techniques using MDA technology and pyrosequencing was used to identify the cell-specific mutations for each of the 3cell lines.
Researchers found that after both experiments, the QIAscout isolated the targeted single cell of interest 97% or 98% of the time using morphology or fluorescence as the selection criteria, respectively. In contrast, the pipetting and serial dilution methods were unable to isolate the single cell of interest for either selection criteria. Both the pipetting and serial dilution methods led to tubes that contained single cells of one of the 3 cell lines – not only single cells of the cell line of interest. In addition, a high number of tubes contained multiple cells (>1 cell) or no cell at all. The QIAscout method shows a highly efficient and reliable new method to visually select cells of interest using common selection criteria, while remaining affordable and easy to use.
Another important thing to note about the QIAscout system is that the mechanism is gentle on cells, making it possible to isolate viable single cells for analysis or clonal expansion. We will talk more about cell viability, contamination and cultivation in an upcoming post!
You can read more details and in-depth experimental steps in the application note: Using the QIAscout for efficient and targeted isolation of single cells selected based on their morphology and fluorescence
For any questions or inquiries about the QIAscout, please contact us!
- 1. Gross, A., Schoendube, J., Zimmermann, S., Steeb, M., Zengerle, R., and Koltay, P. (2015) Technologies for single-cell isolation. Int. J. Mol. Sci. 16, 16897–16919. Link
2. Hu, P., Zhang, W., Xin, H., & Deng, G. (2016). Single cell isolation and analysis. Front. Cell Dev. Biol. 4, 116. Link