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Apr 15, 2009 (Vol. 29, No. 8)

Fine-Tuning Sample Preparation Techniques

Producing High-Quality Data through the Optimization of DNA and RNA Extraction

  • Magnetic Beads

    Click Image To Enlarge +
    Advanced Liquid Logic has developed a digital microfluidics technology to streamline liquid-handling operations.

    Digital microfluidics is a relatively new approach to liquid handling. Discrete droplets are manipulated using electrodes to independently control each droplet. This technology enables extremely flexible lab-on-a-chip devices that can be configured in software to execute virtually any assay protocol.

    “The basic idea behind digital microfluidics is that any liquid handling can be broken down to a specific set of basic operations such as dispense, transport, merge, and split,” explains Vamsee Pamula, Ph.D., CTO at Advanced Liquid Logic “Sequences of basic droplet operations can be combined to create complex liquid-handling protocols. Hundreds of droplets can be simultaneously and independently manipulated, allowing even complex assays to be implemented quickly and reliably."

    Dr. Pamula and his cofounder, Michael Pollack, Ph.D., developed the digital microfluidics technology during their graduate and post-doctoral studies at Duke University. “We have implemented major types of assays including DNA amplification, immunoassays, and enzymatic assays on our digital microfluidic platform because, ultimately, all these assay protocols are just liquid-handling operations that can be broken down into the basic droplet operations,” continues Dr. Pamula.

    Dr. Pamula will be presenting recent results of sample preparation using magnetic beads. “Adding magnetic beads to the platform expands the functionality by automating even complicated operations such as DNA extraction. One way to think of this technology is that the functionality of a liquid-handling robot is built-in within the disposable cartridge, which is controlled electronically through software without any external pumps or valves. It’s inexpensive and integrated.”

    Dr. Pamula notes that the company recently developed a compact benchtop analyzer that is capable of performing enzymatic assays, immunoassays, and DNA amplification. It is currently being evaluated by key partners. The implementation of other assay formats, as well as a portable analyzer is under way.

  • TU-Tagging

    TU-Tagging is cell type-specific analysis of gene expression in vivo and in vitro that uses the uracil phosphoribosyltransferase (UPRT) gene of the protozoan parasite, Toxoplasma gondii, to convert the modified uracil 4-thiouracil into 4-thio-UMP for subsequent incorporation into mRNA.

    Mike Cleary, Ph.D., assistant professor, University of California, Merced School of Medicine, explains that he developed this methodology as a graduate student. “You won’t find UPRT activity in multicellular animals, and thio-containing nucleotides don’t naturally occur in eukaryotic mRNAs, so selective purification of 4TU-tagged transcripts is possible.”

    What Dr. Cleary found is that the thio-substituted compound 2,4-dithiouracil  could be converted into a form that could connect to RNA. “If you start the pulse at 30 minutes, all RNA transcribed at that time will incorporate the TU Tag,” he explains. “I have shown that this works in UPRT-transgenic mouse models and can also be used to selectively tag T. gondii RNA during a mouse infection.”

    Dr. Cleary also recently developed TU-tagging in a multicellular organism, the fruit fly, Drosophila melanogaster. He added that experiments in UPRT-transgenic flies “have demonstrated that TU-tagging can be used to purify cell type specific mRNAs under in vivo conditions with high sensitivity and specificity.”
    Dr. Cleary recently received a California Institute of Medicine grant to further develop the TU-tagging technique by applying it to mammalian tissue culture cell lines and embryonic stem cell lines, and the mouse—two model systems that have proven useful for studying stem cell biology.

    “As an example, this technique could allow stem cell-specific gene expression during interactions between stem cells and niche cells, analysis of gene expression in specific sub-populations of cells that arise during differentiation, identification of genes that are specifically expressed in stem, progenitor, and differentiated cells in vivo, and analysis of gene expression in cancer stem cells in vivo,” concludes Dr. Cleary. “TU-tagging could be an important part of the genomic toolkit. We’re still in the early phases, but there’s a good bit of preliminary data.”


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