By Ben Butkus
Stanford University researchers have published a method for using Fluidigm's digital PCR platform to conduct single-cell, real-time PCR to compare gene expression patterns of single cells.
The protocol is indicative of the increased use of Fluidigm's products for single-cell biology, an application area that the company has been heavily promoting over the past year.
In addition, the method may provide a powerful tool for understanding gene expression and differentiation in induced pluripotent stem cells and human embryonic stem cells for regenerative medicine, the researchers said.
In a paper published last week in Nature Protocols, researchers led by Joseph Wu, associate professor of medicine, cardiology, and radiology at Stanford University School of Medicine, described how they used Fluidigm's BioMark HD platform and Dynamic Array chips to analyze gene expression profiles of single iPSCs or hESCs approximately 11 hours after collection.
The team decided to publish the method after using it to conduct a study published last March in the Journal of Clinical Investigation that demonstrated how single-cell transcriptional profiling revealed heterogeneity in human iPSCs. "A lot of people asked us after that JCI paper how we exactly do this, so we decided to write a detailed protocol," Wu told PCR Insider.
Wu and colleagues began using Fluidigm's platform through the laboratory of fellow Stanford scientist Stephen Quake, a co-founder of the company and a co-author on the recent Nature Protocols paper.
According to Veronica Sanchez-Freire, a postdoc in Wu's lab and also a co-author on the paper, the group needed a tool to compare gene expression between individual cells in single colonies of iPSCs or hESCs with high sensitivity using a limiting amount of sample.
"We were interested in seeing how different gene expression could be in the cell depending on its position in the colony," Sanchez-Freire said. "We are looking at these iPS cells from different cell types and donors, and we always compare them to [human embryonic] stem cells, the gold standard — but we also wanted to see how similar they are [to each other]."
Most traditional gene expression studies, using, for example, quantitative real-time PCR, extract RNA from a large population of cells for downstream expression analysis. "And we saw that when you do that, iPS cells and stem cells are very similar," Sanchez-Freire said. "But when you go to the single-cell level, we saw how the iPS cells are more heterogeneous than the ES cells."
Further, one of the research group's activities has involved injecting stem cells into the hearts of laboratory animals, and then recovering the cells after a certain amount of time to analyze gene expression and differentiation.
"We just want to recover some specific type of cell, or ones that express [certain] genes," Sanchez-Freire said. "In any case, we have very few cells. For us it would be very difficult with that small amount of cells to isolate total RNA and then do real-time PCR, and in some cases, that would not be possible. But with this technique, we can get very small amount of cells and produce that data."
As detailed in their paper, the researchers conducted their experiments with Fluidigm 48.48 Dynamic Array integrated fluidic circuits and the BioMark HD reader, the researchers were able to study the expression of up to 48 genes simultaneously, including at least one housekeeping gene for normalization.
"In each of these chambers, a single real-time qPCR reaction takes place," the researchers wrote. "Dynamic Array IFCs offer the opportunity to combine different standard reagents, an advantage that makes the assay configuration more flexible. The protocol we detail here is intended for the use of TaqMan primers. Alternatively, DNA-binding dyes such as EvaGreen [from Biotium] may be used, allow¬ing for a more affordable selection of real-time PCR primers."
The researchers noted that a key step prior to analysis on the Fluidigm platform is reverse transcription and specific target amplification in a thermal cycler suitable for 96-well plates. This is primarily because the amounts of RNA present in a single cell are on the order of picograms, and can be as low as one to 10 molecules for rare mRNA species of interest, the authors noted.
"When we are using only one cell, we are going down to picograms of RNA," Sanchez-Freire said. "That step is necessary to amplify that small amount of RNA. Some microRNA protocols, because they are very small and not very abundant, also have this kind of pre-amplification step before going to real-time PCR."
One of the main limitations of their method, the researchers noted, was the need to retrieve a much larger number of cells when working with highly heterogeneous cell populations, in order to be confident that the resulting data is representative of the population.
Using their method, the researchers have observed significant heterogeneity at the single-cell level for iPSCs, which they say may account for their less-consistent cardiac and endothelial cell differentiation efficiency, as well as a slower proliferation rate in vivo.
"This may help explain why, in iPS cells, there is just so much more variability when you try to culture them, when you try to differentiate them in a different lineage," Wu said. "One line may work, and another may not work. Even the same colonies in the same patient may have varying abilities to differentiate into certain cell types."
The Stanford group's work is yet another example of the increasing use of Fluidigm's products for single-cell biology studies, an area that the company has been heavily targeting.
For instance, in a November conference call recapping the company's third-quarter 2011 financial results, Fluidigm CEO Gajus Worthington cited several examples of customers using the BioMark system for such work; and said that half of the instruments sold in Q3 and Q2 were destined for such research (PCR Insider, 11/10/2011). Similarly, the company said earlier this year that about 45 percent of BioMark platforms sold in Q4 were for single-cell applications (PCR Insider, 2/16/2012).
Fluidigm also has an ongoing partnership with BD Biosciences to co-host a series of public seminars on isolating and analyzing single cells using a combination of the companies' technologies, including BD's flow cytometry platforms (PCR Insider, 8/4/2011).
However, it is fair to say that a significant portion of single-cell work on the BioMark platform is taking place through the Quake connection at Stanford. For example, Stanford University spinout Quanticel Pharmaceuticals — which is conducting single-cell genomic analysis for drug discovery and has signed a $45 million deal with Celgene for that purpose – is a BioMark user. And in January, another Stanford group published a Nature Protocols paper describing the use of the BioMark platform for profiling gene expression in single neuronal cells (PCR Insider, 1/5/2012).
Wu said this week that although his group began its work with the Quake lab's platform, "right now I think there are five or six [BioMark] machines on the entire [Stanford] campus right now."
"We have pretty big lab, and it’s a pretty heavily subscribed system to look at different questions," Wu added.
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