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Mar 1, 2009 (Vol. 29, No. 5)

Quality Makes a Difference in RNA Prep

New Methodologies Are Being Counted On to Bring Order and Repeatability to Gene Expression

  • Click Image To Enlarge +
    Sections of tissue, before (left) and after (middle) laser microdissection using the Leica AS LMD System, and an image of dissected material in the collection tube using inspection mode (right)

    In recent years, drug discovery has become a search for targets that take the form of gene-expression differences between normal and disease states. One of the leading approaches has been based on building gene-expression profiles derived from microarray analysis. In such investigations RNA samples from different tissues, organs, disease states, and individuals are evaluated against microarrays consisting of large numbers of gene fragments. While this strategy is widely used, initially results were inconsistent, they could not be duplicated, and putative markers failed to survive critical assessment.

    One reason for the failure of such studies was the poor quality of the RNA preparations, given that this molecule is easily degradable and stringent precautions must be met in its isolation. But recently, a number of suppliers have developed approaches to ensure high-quality RNA isolation.

    Qiagen offers a variety of products for the molecular biologist, including kits to ease the challenges of RNA purification, according to Barry Westfall, regional marketing manager for North America. Qiagen’s purification kit for nucleic acids and proteins goes by the trade name AllPrep, which includes the stabilization reagent, also available as a free-standing product.

    The RNeasy Mini Kit was designed to process up to 100 µg total RNA from mammalian cells, tissues, and yeast. These kits are based on the use of disposable modules with silica membranes, which become attractive to the nucleic acids because of the buffer systems.

    All the companies surveyed for this article offer alternatives to the phenol/chloroform extraction procedure, a venerable warhorse whose use goes back to the early days of molecular biology in the 1960s. Today there is great pressure on both academic and commercial laboratories to lower their contribution to the contamination of the water supply to zero.

    However, the principle driver for new methodologies has been the need for a technique that could deliver high-quality total RNA in minutes. Moreover, since researchers aim to analyze samples smaller by orders of magnitude than those that they would have attacked 20 years ago, Qiagen has designed technologies that can derive RNA, DNA, and protein all from the same microsample.

    This reconfiguration means that Qiagen can now deliver products that generate ready-to-use RNA in yields from very small to large amounts of starting material. Their miRNeasy and miScript products allow preservation of all the miRNA in a sample. Thus the RNA molecules are all purified and material can be scanned when a new miRNA is discovered. 

    Qiagen also offers a number of robotic equipment options to move processing of samples forward at an accelerated pace. The QIAsymphony SP is a compact integrated system, with built-in touchscreen and computer designed to operate an automated system with flexible processing of a wide range of samples. It can handle up to 96 samples, in batches of up to 24 per run, with sample volumes up to 1 mL.

    The system is designed for automated purification of nucleic acids or 6xHis-tagged recombinant proteins from a range of sample types. The platform is easy-to-use with prefilled reagent cartridges that permit flexible processing of 1 to 96 samples per run. The use of bar code reading enables full sample and reagent tracking. An additional product is FastLane, allowing qPCR straight from cells, including SYBR Green, Probe, or Multiplex probe qPCR.

    In addition, Qiagen has embarked upon a partnership with Leica Microsystems to produce a laser microdissection and nucleic acid purification tool. The system can cut discrete portions of a tissue section with great precision; the excised portion is collected by gravity feed into a tube and can then be analyzed for specific DNA or RNA content. The technique favors the use of flash-frozen cryosections to avoid formalin and produce the highest quality nucleic acid molecules.

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