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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

  • Isolation and Purification

    Another company looking at optimization of DNA and RNA extraction as enabling personalized medicine is AROS  Applied Biotechnology. “We have developed a procedure for isolation of microRNA and genomic DNA in addition to total RNA from whole blood stabilized in PAXgene Blood RNA tubes,” says Mogens Kruhøffer, Ph.D., CSO. “This procedure is based on automatic extraction on a BioRobot MDx and includes isolation of DNA from a fraction of the stabilized blood and recovery of small RNA species that are otherwise lost.”

    Dr. Kruhøffer notes that its methodology is suitable for large-scale experiments, and is also amenable to further automation. “Procured total RNA and DNA was tested using Affymetrix Expression and single-nucleotide polymorphism GeneChips, respectively, and isolated microRNA was tested using spotted locked nucleic acid-based microarrays,” he explains. “Consequently, the yield and quality of total RNA, microRNA, and DNA from a single PAXgene Blood RNA tube is sufficient for downstream microarray analysis.”

    Dr. Kruhøffer will also be discussing his company’s part in the standardization and improvement of generic preanalytical tools and procedures for in vitro diagnostics, or SPIDIA for short. This initiative, launched late last year, seeks to expand the potential and utility of in vitro diagnostics through the creation of new standards, and preanalytical tools and procedures such as the collection, handling, and processing of blood, tissue, tumor, and other sample materials. “When there is variation in blood samples, we need to know why—are they properly stabilized?” says Dr. Kruhøffer. “Creating standards that everyone adheres to is crucial.”

    “There are many advantages to working with FFPE,” comments Carlos Moreno, Ph.D, assistant professor of pathology, Emory University School of Medicine. “Some of the advantages are excellent clinical annotations, particularly for those from clinical trials, and any discoveries made in FFPE are potentially applicable for clinical translation.”

  • RNA Extraction from FFPE Tissues

    “What we have set out to do is to develop a methodology for biomarker analysis of DNA and RNA geared toward using the Illumina platform. We have moved toward doing this in an automated high-throughput manner, using a 96-well format. We have adapted the Ambion—now part of Life Technologies—RecoverAll™ kit for FFPE using a magnetic bead strategy. We have been using the MagMax 96 Flex instrument with a MagMax back end technology for RNA extraction. This has enabled extraction of high-quality RNA from FFPE samples, including microRNAs.”

    “The real bottleneck in doing the extractions,” Dr. Moreno continues, “is the de-parafinization and protease digestion steps. We use multichannel pipettors, but the deparrafinization step is not amenable to automation and it’s limiting in throughput. Once we have the lysates prepared, however, we can load it onto the MagMax 96 Flex robot and do a large number of samples fairly quickly.”

    Dr. Moreno says that his group is involved in analysis of three large clinical trials, and for each will be looking at different biomarkers for use in personalized medicine applications that might be prognostic or predictive in nature.

    “A large advantage to what we are doing is that we are tracking these samples in a laboratory information system (LIMS) in a CLIA-certified laboratory. This can help translate our biomarker discoveries quickly into a form that can be adapted for clinical use. We are trying to leverage the advantages of working in a CLIA environment, using standard operating procedures, protocols, LIMS, and bar codes—all these things help us with data management and data analysis. And all of these things are critical in processing and profiling thousands of samples.”

    “Eventually,” he continues, “we’d like to take these assays and turn them into clinically useful, readily available biomarkers that can be adopted by any clinical pathology lab that can run real-time PCRs. Ultimately, you want to improve clinical outcomes; these personalized biomarkers will, hopefully, help guide patient care in the future.”

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