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May 1, 2010 (Vol. 30, No. 9)

Nucleic Acid Sample Prep

Faster, Highly Automated Methods Are Being Developed

  • A common theme of many of the presentations at the Knowledge Foundation’s “Sample Prep” conference, to be held this month in Baltimore, will be how to purify nucleic acids, including DNA, RNA, and miRNA, from different types of samples using a sensitive and reproducible method that can be performed in as automated a fashion as possible.

    Whether these isolation procedures are performed in a research setting, a clinical diagnostic laboratory, or a field or military setting, the goal is to be able to load a sample and the necessary reagents and buffers into an instrument, press a button, and output the pure nucleic acid component for subsequent identification and quantification.

    Two main goals drove the development of Norgen Biotek’s nucleic acid sample-preparation technology, which is based on a proprietary resin/matrix for purifying DNA, RNA, and proteins—the ability to isolate total DNA, including miRNA without the use of phenol and the ability to modify this technology for use in resource-limited areas.

    To achieve its first goal, Norgen developed a nucleic acid purification strategy that does not require phenol or chloroform. This approach can isolate circulating total RNA from various sample types including plasma, serum, or blood with the sensitivity and reproducibility needed for use in diagnostic applications.

    Studies aimed at correlating up- and downregulation of specific miRNAs with the presence of cancer in humans are ongoing, and at the meeting, Yousef Haj-Ahmad, Ph.D., president and CEO, will describe a research project that focuses on prostate cancer.

    These types of studies have been hindered by the high degree of sample-to-sample variability and the need to repeat miRNA measurements in each individual sample at least three times. Dr. Haj-Ahmad says that Norgen’s extraction procedure yields a consistent result that can facilitate the development and use of miRNA biomarkers in cancer research.

    Norgen has transitioned its nucleic acid purification technology into the diagnostic market with the launch of more than 30 kits—initially available for research use—for applications such as surveillance of drug-resistant pathogens, field surveillance, and epidemiologic studies. The company’s kits target commonly transmitted diseases such as HPV, HIV, herpes simplex virus-1 and -2, chlamydia, and gonorrhea, as well as hepatitis B virus and food-borne pathogens.

    Another key feature of Norgen’s technology is its ease of adaptability for use in resource-limited areas due to the properties of the company’s resin, Dr. Haj-Ahmad says. Inhibitor-free nucleic acid isolation from urine samples does not require laboratory equipment such as refrigerators or centrifuges. Extraction involves collection of a urine sample in a tube containing resin to concentrate the analyte of interest. The analyte binds to the resin on mixing, and due to the high density of the resin, the complex falls to the bottom of the tube.

    The urine is then decanted, the resin is washed with 1–2 mL of buffer to remove contaminants, and the analyte is then eluted at a 50- to 100-fold increased concentration compared to the original sample. The analyte can then be detected quantitatively or qualitatively depending on the detection method.

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