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Feature Articles : Apr 15, 2009 (Vol. 29, No. 8)

Fine-Tuning Sample Preparation Techniques

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

The push toward personalized medicine and the compression of the drug development pipeline puts more pressure than ever on the genome space. At CHI’s “Genomics Tools & Technologies” meeting to be held in June, the ever-changing and challenging issues around extracting, creating, and keeping samples and templates of highest quality will be addressed, as this is the key factor for producing high-throughput data of optimal quality.

“There are quite a few challenges,” comments Austin Tanney, Ph.D., scientific liaison manager, Almac Diagnostics . “But there are a number of really good reasons to work in this space, one of the chief reasons being what this can mean for personalized medicine.”

FFPE Tissue

FFPE tissues will be a hot topic at this conference. “One of the reasons we work with FFPE is that there are literally hundreds of thousands of samples available—a huge amount of information to work with,” says Dr. Tanney. “Doing retrospective studies can cut off years of development time for a diagnostic. What is most important, however, is that if a diagnostic test works from FFPE there is no required change to clinical practice to use this test. This is not a case when the test must be carried out on fresh frozen material.”

Personalized medicine, in this case, using genomics to determine a course of care, is still in its infancy, but advances in technology to enable DNA and RNA extraction are advancing the field. Dr. Tanney’s talk will discuss the ways in which his group has been able to use what they have found in FFPE samples to advance Almac’s cancer studies. “Cancer has always had a one-size-fits-all treatment regimen, even though it’s a heterogeneous disease,” continues Dr. Tanney. “Clearly, this is an area where treatment can be personalized.”

But working with FFPE has its challenges, he notes. “High-quality RNA can easily be reverse transcribed and amplified, and in degraded RNA, use of oligo dT primers can be a limiting step. Getting usable RNA is also a challenge because cross-links get broken—extracting RNA degrades it.”

To meet the challenges of the complexity of working with FFPE tissues in the cancer field, Almac developed Cancer DSA research tools, which are high-density microarrays based on the transcriptome of an individual disease and are capable of obtaining robust data from FFPE samples and delivering additional information on the chosen disease setting.

“One of the biggest issues in developing prognostic and predictive tests is the availability of samples,” points out Dr. Tanney. “Fresh frozen is virtually impossible, but working with FFPE is not easy, either.”

And working with a disease as heterogenous as cancer is akin to trying to hit a moving target. “For example, there are at least five subgroups of breast cancer,” he adds. “That there is such diversity in the disease and not in treatment, means that there has to be better ways of treating cancer than the way we are presently going about it.”

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.”

Magnetic Beads

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.”