Depleting the Ozone: Distinguishing the Differences
Is it possible that gases in the air that protect us from the sun could combine with pollutants and strongly affect microarray data?
Scientists in Pat Brown's laboratory at Stanford University School of Medicine together with SciGene have been working collaboratively to address this issue. While it is generally accepted that nominal ozone levels degrade signals from Cy5 and other fluorescent dyes used in microarray applications, it has never been absolutely quantified until now. Brown's' team empirically tested the effects of different levels of ozone on both the signal in the Cy5 channel as well as the Cy5/Cy3 ratio.
"Ozone levels affect the Cy5 signal disproportionately more than the Cy3 signal. Most labs use a Cy5/Cy3 ratio on their oligo or cDNA array, so if ozone is reducing the signal in one of the channels but not the other, it's essentially changing the ratio and thereby significantly altering your biological data by an artifactual effect," says Kate Rubins, a post-doctoral researcher at Stanford.
Since ozone is atmospheric, it is very hard to monitor under controlled experimental conditions. For this reason, SciGene developed the NoZone Workspace, a sealed enclosure with a highly efficient ozone filtration system, including an ozone scrubber and monitor that maintain internal ozone levels under 5 parts per billion (ppb). The NoZone Ozone Scrubber pumps lab air through a bed of catalysts that convert ozone to oxygen before it enters the enclosure. "The positive pressure of reduced ozone air created within the enclosure ensures that low ozone levels are maintained," says Jim Stanchfield, president and CEO of SciGene.
As it is neither practical nor financially feasible for laboratories to invest in a complete ozone-free building, SciGene's BriteSpot Microarray Workstation, including the NoZone Workspace, the Hybex Microarray Incubation System, and The Little Dipper, gives researchers a complete system for incubating and processing microarrays in an ozone-safe environment.
According to Rubins, the NoZone Workspace allowed her team to deplete all of the ozone in the chamber. This served as their positive control: < 5 ppb conditions. "We did several sets of replicate arrays under 5 ppb of ozone, scanned them, and took the signal on those arrays as our 100 percent," says Rubins. Using an ozone generator, they increased the ozone levels to 30-60 ppb—typical atmospheric ozone levels in most labs—for one of the tests. They also examined a higher ozone level range of 80-90 ppb. All arrays were scanned using the same laser settings, and the percentage of signal in the higher-ozone conditions was compared with the no-ozone conditions.
"We saw a loss in the Cy5 signal as you increased the ozone. At 30-60 ppb, we saw a signal loss of approximately 40 percent, and even more so, approximately 50 percent with 80-90 ppb," says Rubins. This is not a huge issue if you are only running 24 arrays in an experiment and can get them all done on the same day, because the signal loss is uniform. The big problem comes when you have an array project of ~100 arrays and you are doing them on different days and the ozone levels/conditions vary on different days.
One of the more surprising results—not yet reported by any other laboratory—is that the effect of the ozone degradation on the Cy5 signal across the array is not uniform. This point was emphasized by both Stanchfield and Rubins as potentially disastrous.
They reported a left-to-right red gradient—or right-to-left depending on how the arrays are positioned vertically in the rack—during the washing and drying step. "So the signal on the left side of the array will start out bright red and decrease to almost nothing by the time you get to the right side of the array," says Rubins.
The results were derived by printing an array in 48 (4x12) sectors, composed of about 40,000 spots. They looked at the signal in each of these sectors in a single array. "What we found was at 30-60 ppb, the signal on the left side almost exactly matched the 0 ppb conditions—very bright red—but as you reached sector 4 on the right side of the array, the signal had lost about 40 percent of its signal," says Rubins.
By quantifying this left-to-right red gradient phenomenon, they were able to conclude that this difference in signal was not due to background noise but rather to the actual decreased red signal on the array spots.
This phenomenon holds true when utilizing both one-color or two-color scans. Brown's group says they are not certain if this holds true for Affymetrix arrays, but is definitely the case when employing long oligo and spotted arrays.
"The result of the left-to-right gradient differences will significantly distort your microarray data. For example, a gene that happened to be placed on the left side of the array will have a completely different ratio readout than that from a gene that happened to be placed on the right side," says Rubins.
This can especially be a problem, for example, if you are studying cancer and have a list of 10 genes that discriminate different subtypes of cancer. If half are on one side of the array and half are on the other, you are going to get completely erroneous results.
"The more carefully people can control these sources of data, the more fidelity they will have between replicate experiments and the better their RT-PCR data will match their microarray data. It's a case for validation of small numbers of targets," says Rubins.
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2nd Annual Electronic Laboratory Notebooks - February 27-28,Philadelphia, PA
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CBI's Drug Development – From Animal Models to Phase I; Pursue Quality Leads by Expediting PK/PD and Efficacy Data Collection -- April 10 - 11, 2006, Philadelphia, PA
Gene Therapy and Stem Cells Week, March 8 – 10, 2006, Cambridge, MA
Life Sciences Conference + Expo - April 3 - 5, Boston, MA
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Exon-for-Exon: MEEBOChip and HEEBOChip Tell All
A collaboration of researchers from Stanford University School of Medicine, UCSF, Stowers Institute for Medical Research, Rockefeller University, and Basel University, led by Ash Alizadeh from Stanford, have designed and built an open-source Mouse Exonic Evidence-Based Oligonucleotide Chip (MEEBOChip) and the human counterpart, HEEBOChip.
Both arrays are based on a novel selection of 70-mer exonic long oligos from a genomic annotation of the corresponding complete genomic sequences, using a transcriptome-based annotation of exon structure for each genomic locus. Using a combination of existing and custom-tailored tools and datasets, the group built and performed a systematic exam of transcript-supported exon structure for each genomic locus at the base pair level.
Why exon-based? According to the researchers, this strategy allowed them to select both constitutive and alterative exons for nearly every gene in the corresponding genome allowing unparalleled exploration of mouse and human biology. It also enables the recognition of the same continuous sequence in RNA transcripts, and in genomic DNA, which is useful for expression in analysis and CGH.
The arrays that were used in the ozone depletion experiments in Pat Brown's lab at Stanford University (see above) were MEEBOChip arrays. They are currently being printed by, and can be purchased through, the Stanford Functional Genomics Facility.
News in Brief
The National Human Genome Research Institute (NHGRI) has purchased a 15-seat license of the VizX Labs' Genesifter Core Edition online microarray data analysis system for use in the NHGRI Computational Genomics Unit.
SCHOTT has secured a $1 million grant for the development of a new microarray slide that will be used in an automated system for detecting the presence of biological agents.
Applied Biosystems announced that its Advanced Gene Expression Service Provider Program has expanded globally with the addition of 11 microarray service provider centers throughout North America, Europe, and Asia. These include Bionomics Research and Technology Center at the University of Medicine and Dentistry of New Jersey and Rutgers University; Geneservice Limited; Genome Express; IMGM Laboratories; Labindia; Macrogen; Center for Medical Research, Medical University Graz; Ming Shin Biotech; Trinity College Dublin; Norwegian Microarray Consortium; University of Bergen; and Vanderbilt Shared Microarray Resource. They will offer Applied Biosystems microarray gene expression tools and services, including microarrays for human, mouse, and rat genes to enhance service providers' business offerings.
Helix BioPharma Corp. received a milestone payment from Lumera Corporation under the terms of the license agreement to develop a high-throughput, label-free detection platform in collaboration with the Institute for Systems Biology. The platform may enable biologists to isolate protein samples for testing and analysis. Lumera is developing specialized microarrays combining Helix's Heterodimer Protein technology and Lumera's nanosurface modification chemistry, which will allow researchers to consistently take an existing DNA array to produce protein arrays that accurately mimic the native living cell environment of the body.
Product Spotlight
Ariadne Genomics has released Pathway Studio 4.0 pathway analysis software, which contains tools for microarray data analysis and pathway dynamics simulation, as well as over 1,000 reconstructed pathways. It includes a pathway simulation module to explore the dynamic behavior of pathways, tools for building relevance networks from the gene expression data, and Ariadne's MedScan Reader biomedical literature browser.
Nexterion, a division of SCHOTT North America, has developed HiSens slides designed to enhance the signal-to-noise ratios from microarray experiments by over eight times. According to the company, these slides are well suited to applications requiring low concentrations of target material or which cannot be reliably amplified, such as measuring mRNAs or low abundant proteins.
Ingenuity Systems' new Ingenuity Pathways Analysis (IPA) 3.1, a Web-based software application that helps biologists and bioinformaticians understand biological mechanisms, pathways, and functions. IPA 3.1 integrates with third-party and customer applications through dedicated Application Programming Interfaces and supports Affymetrix GeneChip Human Mapping 10K, 100K, and 500K Array sets. Many of the new features include mapping files for SNPs, and updated content containing 1.4 million biological findings.
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Upcoming Industry Awards
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Bio-IT World Best Practices - Call for Entries - Early Bird Deadline February 28th
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CBI's Drug Development – From Animal Models to Phase I; Pursue Quality Leads by Expediting PK/PD and Efficacy Data Collection -- April 10 - 11, 2006, Philadelphia, PA
Expediting drug development is a major concern for the pharmaceutical industry. Maximizing preclinical and early clinical development not only speeds up the process, but also reduces money wasted on pursuing compounds with little or no potential for approval. The purpose of this event is to bridge the gap between preclinical and Phase I development. High level speakers present crucial solutions ranging from developing new humanized transgenic animal models to designing more efficient Phase I trials. For more information or to register, please contact the Center for Business Intelligence toll free by phone at 1-800-817-8601 or via e-mail at [email protected]. Visit: https://www.cbinet.com/show_conference.cfm?confCode=HB637
Gene Therapy and Stem Cells Week, March 8 – 10, 2006, Cambridge, MA
CBI's 2nd Conference on Commercializing Stem Cells for Therapeutic Applications, March 9-10, 2006, examines following key issues: - Stem Cell Sourcing and Utility for Therapeutic Applications; - Business Models, Funding Challenges and IP Strategies for the Commercialization of Stem Cells; - Legal, Political and Ethical Issues Affecting Stem Cell Research; - Update on Stem Cells in Cardiac Clinical Trials.Plus! Attend a One-Day Symposium on Gene Therapy Research on March 8, 2006. Register for all three days with special package pricing. For more information or to register, please contact the Center for Business Intelligence toll free by phone at 1-800-817-8601 or visit https://www.cbinet.com/show_conference.cfm?confCode=HB630
Life Sciences Conference + Expo - April 3 - 5, Boston, MA
The Life Sciences Conference + Expo features an outstanding program of keynotes, three-day conference tracks, educational workshops, exhibit floor, and award programs and special events. Speakers will discuss the latest technology developments and research breakthroughs on the complete spectrum of topics involved in drug discovery through market delivery. Learn from actual case studies and panel presentations on how the leading pharma and biotech companies utilize IT and informatics to enhance drug process and speed the time to market.
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