By Ben Butkus
The amplification of nucleic acids from single cells was a recurring theme at Cambridge Healthtech Institute's XGen Congress, held last week in San Diego.
Scientists from Hitachi, the Institute for Systems Biology, and the J. Craig Venter Institute all gave presentations on the single-cell amplification methods being developed in their labs.
The technology to perform the methods has been around for several years, but scientists are just beginning to understand how it can be applied to niche research areas.
In general, the researchers who presented at XGen are using single-cell amplification techniques in situations where averaging gene expression from many cells or tissues might mask important individual variations, such as in cancer stem cell research. In addition, the technique is being explored as a way to prepare DNA for subsequent downstream sequencing studies of individual cells.
Hideki Kambara, a fellow at Hitachi's Central Research Laboratory in Tokyo, discussed a method under development in his lab that couples qPCR with reusable cDNA libraries to analyze the expression of multiple genes in single cells without pre-amplification.
In this method, single cells are isolated, lysed, and treated with DNAse in a sample tube. Then, using magnetic beads, all mRNA is converted to cDNA, which is held on the beads and removed as supernatant from the sample. This provides a reusable cDNA library sample that can serve as a template for subsequent real-time qPCR analysis on individual genes from the library.
Advantages of the method include the fact that it does not require pre-amplification, which can introduce PCR bias; that additional analysis is possible with reusable cDNA libraries; and that it is cost effective compared with other PCR methods.
Kambara also outlined some key points that need to be addressed to improve the method. For instance, to avoid adsorption of mRNA or cDNA on various surfaces, particularly the reaction chamber, Kambara's lab uses an MPC polymer to coat the surface of the chamber and uses one reaction tube through the experiment.
In addition, Kambara said that selecting the appropriate reverse transcriptase is a key factor for increasing the efficiency of mRNA capture and conversion to cDNA. The lab has tested several reverse transcriptase kits in its method and found that Clontech's Advantage RT-for-PCR, Life Technologies' ThermoScript RT-PCR, and Roche's Transcriptor first-strand cDNA synthesis kits produced the greatest number of cDNA molecules.
Lastly, cDNA desorption from the magnetic bead surfaces after multiple thermal cycles can compromise the reusability of the cDNA library. To address this issue, Kambara's lab uses low-temperature qPCR by adding formamide, which is effective for reducing primer melting temperature.
Kambara's lab has thus far used the method to analyze 12 single-cell cDNA libraries and demonstrated that the expression levels of four different housekeeping genes fluctuated from cell to cell despite receiving the same treatment. The group has also used the technique to examine the differentiation of mouse mesenchymal stem cells.
In addition, Kambara said that the technology is expected to serve as a stepping stone in the efforts of Hitachi and others to perform massively parallel gene expression profiling of multiple single cells.
Untangling Cell Heterogeneity
Meantime, in another XGen presentation, Adrian Ozinsky of the Institute for Systems Biology described how his lab is using real-time PCR for simultaneous multiplexed gene detection in multiple single-cell samples.
In general, Ozinsky's lab is attempting to develop microfluidic tools to probe the spectrum and influence of cell-cell heterogeneity on immune response by measuring responses at the single-cell level, according to the lab's website.
To do this, they are developing techniques that allow multiple responses to be measured simultaneously in order to define coordinated response patterns from the same individual cell and from multiple cells in parallel.
In their real-time PCR method, Ozinsky's group first uses flow cytometry to sort a controlled number of cells into wells of a 96-well plate. This is followed by cDNA synthesis to produce a cDNA lysate, addition of Taqman qRT-PCR reagents, and12 to 18 cycles of pooled amplification.
All of this allows the researchers to quantify each gene individually – after some data-crunching methods that the group is still working out.
"Doing the experiment is relatively straightforward," Ozinsky said. "But analyzing the data is a bit more complicated."
Despite this, as described in a research article published last summer in PLoS One, Ozinsky and colleagues have assessed the performance of the technique using mRNA and DNA standards and cell samples, and demonstrated a detection sensitivity of about 30 mRNA molecules per cell and a fractional error of 15 percent.
In that paper, they also showed how they used the method to expose unexpected heterogeneity in the expression of five immune-related genes in sets of single macrophages activated by different microbial stimuli.
Additionally, some members of the Ozinsky lab are using single-cell gene expression fingerprints to attempt to identify breast cancer stem cells. In this work, the researchers have added to the mix Fluidigm's BioMark 48.48 microfluidics platform to further multiplex their single-cell measurements in a single run.
That work is ongoing, but Ozinsky said that his lab's work thus far has demonstrated that single-cell mRNA assays are feasible, and that they can be scaled for insight into the behavior of individual cells and cell populations.
Lastly, Roger Lasken of the J. Craig Venter Institute presented a method for amplifying genomic DNA from small samples and single cells using multiple displacement amplification, an alternative to PCR that has been demonstrated to result in better genome coverage than PCR-based methods.
In an interview with PCR Insider following the XGen conference, Lasker said that he developed the MDA technique while he was director of genomics at Molecular Staging, which in 2004 was acquired by Qiagen. Currently the technology is sold by Qiagen under the brand name Repli-g and by GE Healthcare under the brand names TempliPhi and GenomiPhi.
The MDA method is used most often as the basis for further single-cell sequencing studies, and involves annealing random hexamer primers to a template and synthesizing DNA using Φ29 DNA polymerase. The reaction is isothermal and thus performed at a constant temperature of 30º C.
Lasken said that the technique provides "the lowest amplification bias" of any current nucleic acid amplification method. His group is currently using MDA at JCVI to conduct single-cell sequencing of uncultured microbes from the human microbiome.
"If you can sequence from a single cell then you don't need to culture bacteria," Lasken said, adding that many important bacteria, especially those found in the human body, have not yet been sequenced.
"Many of these are difficult to culture — some may require that community of bacteria to thrive, and some require very specialized media to culture," he added. "A lot of labs are now using this method because it's really the only way to get access to some of these bacteria … and produce at least a draft sequence."