Comprehensive Profiling of miRNAs in Stem Cells utilizing Microfluidics Technology
By BiotechDaily International staff writers
Posted on 28 Oct 2009
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| | Image: Colored scanning electron micrograph (SEM) of stem cells (Photo courtesy of SPL). |
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MicroRNAs (miRNAs) have been shown to be key regulators of hematopoiesis. However, the detailed profile of miRNA expression throughout hematopoietic development has not been charted, due mostly to difficulties in obtaining highly purified samples of stem and progenitor cells as well as profiling hundreds of miRNAs from low cell numbers. To overcome these limitations, investigators have demonstrated how microfluidic technologies were combined with a 288-plex real time polymerase chain reaction (PCR) approach, allowing the detection of 288 miRNAs in small numbers (c. 3000) of cells.
The researchers, from the BC Cancer Agency (Vancouver, Canada) and University of British Columbia (Vancouver, Canada), revealed how over 20 unique murine hematopoietic cell populations were isolated through fluorescence activated cell sorting (FACS), including hematopoietic stem cells (HSCs), myeloid and lymphoid progenitor cells, and mature lineage populations. Several thousand nanoliter volume quantitative PCR reactions were performed using Fluidigm (South San Francisco, CA, USA) BioMark 48.48 Dynamic Arrays and miRNA-specific TaqMan probes, developed by Applied Biosystems (Brisbane, CA, USA). For each miRNA, synthetic miRNA standards were used to determine the absolute number of miRNA molecules per cell; 133 of the 288 miRNAs tested were detected across all cell populations.
The researchers concluded that Fluidigm’s dynamic array provides a rapid, sensitive, and cost-effective method for miRNA profiling in rare cell populations. The sensitivity of Fluidigm’s dynamic array is shown to detect heterogeneity in miRNA expression at the single cell level in purified populations.
The Fluidigm BioMark system uses integrated fluidic circuit technology to reduce complexity, improve throughput with nanoliter precision, and provide significant cost-savings (less reagent and smaller samples) for high-throughput PCR applications. Fluidigm’s 48.48 Dynamic Array is capable of performing 2,304 simultaneous real-time PCR experiments running gold-standard TaqMan assays in nanoliter quantities.
Fluidigm develops, manufactures, and markets proprietary integrated fluidic circuit (IFC) systems that significantly improve productivity in life-science research. Fluidigm’s IFCs enable the simultaneous performance of thousands of advanced biochemical measurements in extremely minute volumes. These “integrated circuits for biology” are made possible by miniaturizing and integrating liquid-handling components on a single microfluidic device (IFC). Fluidigm’s systems, consisting of instrumentation, software, and single-use chips, increase throughput, decrease costs, and enhance sensitivity compared to traditional laboratory systems. Fluidigm products have not been cleared or approved by the Food and Drug Administration for use as a diagnostic and are only available for research use.
The research was nominated as best poster at RNAi Europe, September 2009, held in Berlin, Germany.
Related Links:
BC Cancer Agency
University of British Columbia
Fluidigm
Applied Biosystems
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