SOLICITATION NOTICE
R -- Zebrafish Microarray Hybridization
- Notice Date
- 5/10/2005
- Notice Type
- Solicitation Notice
- Contracting Office
- RTP Procurement Operations Division (D143-01) Research Triangle Park, NC 27711
- ZIP Code
- 27711
- Solicitation Number
- PR-NC-05-10395
- Response Due
- 5/24/2005
- Archive Date
- 6/23/2005
- Point of Contact
- BRENDA J. KIMBLE, Contract Specialist, Phone: (919) 541-2897, E-Mail: kimble.joy@epa.gov
- E-Mail Address
-
BRENDA J. KIMBLE
(kimble.joy@epa.gov)
- Small Business Set-Aside
- N/A
- Description
- STATEMENT OF WORK Hybridization of zebrafish RNA samples on Agilent zebrafish microarrays BACKGROUND As part of an Office of Research Development (ORD) wide computational toxicology project, the combination of whole organism endpoints, genomic, proteomic, and metabonomic approaches, and computational modeling will be used in small fish models to identify new molecular biomarkers of exposure to endocrine disrupting compounds (EDCs) and link those biomarkers to effects that are relevant for both diagnostic and predictive risk assessments. Using zebrafish (Danio rerio), flow-through exposures of differing durations will be conducted for 9 chemicals using sexually-mature animals (ab, wild-type strain) from existing on-site cultures. Four replicate tanks containing both males and females will be used for each treatment at Mid-Continent Ecology Division (MED, Duluth, MN). Gonad, brain, and liver samples will be collected from fish at appropriate intervals and preserved in RNA later. Samples will be shipped on wet ice to Ecological Exposure Research Division (EERD, Cincinnati, OH) for RNA isolation. Zebrafish oligonucleotide microarrays and hybridization services will be used to identify genes and pathways that respond to the various chemicals and disrupt the hypothalamus-pituitary-gonad (HPG) axis in adult tissues. This ?mining? approach for identifying key indicators out of over 22,000 potential genes will help to isolate and identify the likely indicators for extrapolation to the fathead minnow as an environmental model. For chemicals where the mode of action (MOA) has been characterized ( -trenbolone, flutamide, vinclozolin, fadrozole, and prochloraz), samples for microarray analyses will be limited initially to early time points of 24 and 48 hours in order to assess the early transcriptomic responses. Although multiple tissues will be dissected from each chemical exposure, single tissues will be assessed via microarray analyses according to ongoing fathead minnow studies and known pathway interactions for each chemical, with the exception of prochloraz which is known to have mixed MOA affecting liver and gonad. Four replicates of pooled RNA from 2 individuals (8 fish) will be used per treatment to increase the statistical power of analyses while helping to reduce variability between individuals. No less than 2 randomly chosen pooled samples will be hybridized on duplicate arrays to assess experimental variability. For a single tissue, 3 doses (control, 2 chemical doses), and 2 time points, a total of 24 arrays will be required with an additional 2 arrays for dye flip/duplicate controls. Prochloraz exposures will be assessed in 2 tissues for a total of 48 arrays with additional arrays for controls. THE CONTRACTOR SHALL PERFORM THE FOLLOWING TASKS: Custom Zebrafish Array Services The contractor shall provide the Environmental Protection Agency (EPA) array services using a custom printed 60-mer 22K Zebrafish oligo array. The contractor shall provide consultation on research goals and experimental design, RNA QC and labeling, array hybridization, image scanning and QC, data extraction, and data analysis. The contractor shall utilize Agilent equipment to determine the quality and concentration of incoming total RNA samples. Reagents for arrays shall be obtained from the same array vendor to ensure QA/QC throughout the contract. Deposition of these data into the National Center for Toxicogenomics (NCT) Chemical Effects in Biological Systems (CEBS) Knowledge Base, whose goal is to support research that promotes mechanistic understanding of environmentally induced toxicity and disease, is an important requirement of this project. Quality assurance and control (QA/QC) are critical in obtaining highly reproducible microarray results. Ensuring quality shall be performef by a highly trained and expert staff. The contractor shall provide EPA with documentation of tested and validated Standard Operating Procedures (SOPs). SOPs shall employ rigorous QC measures at each individual step of the process, starting with the experimental design of the study, and proceeding through labeling of the RNAs, microarray hybridization and scanning, and acquisition of the data. The contractor shall perform the following 2 categories of quality control as follows: Category 1 RNA and Labeling Quality Control - Including the 260/280 ratio, 260/230 ratio, 28S/18S ratio; and cRNA yield. RNA samples shall be delivered in pure nuclease free water, frozen in dry ice, by overnight shipping. Upon receipt of RNA, the Contractor shall run samples through an Agilent Bioanalyzer for analysis of RNA quality. Good quality RNAs with 28S/18S ratios >1.49 and a low baseline between the 18S and Nano Marker (first fluorescent peak before 24 seconds) are considered acceptable. - RNA samples with visible signs of impurities (260/280 or 260/280 less than 2.0) or low concentration are reported to EPA and shall not be further processed without consultation with EPA. Category 2 Process and Procedural Control - For arrays, 1 to 2 ?g of total RNA shall be used for each labeling reaction. EPA will specify which samples should be combined for pooling. Labeling of cRNA, hybridization, washes, and scanning shall be performed according to array vendor optimization, which include spike in controls to assess cRNA production efficiency. After completion of the array hybridizations, scanning and data extraction, Contractor shall QC the images according to the following guidelines: - Image quantitation parameters shall be identical throughout the experiment. - The raw image shall be free from obvious artifacts (bright areas, dark areas, scratches, dust, intensity gradients, smudges, bright edges, streaking, uneven background) and the entire microarray surface shall be visible and in focus. - The raw feature intensities shall range from just above zero to near saturation. That is, the full linear range of the scanner shall be exploited. In general, the median shall be as high as possible while simultaneously avoiding significant numbers of saturated spots. - Quality scores for feature quantitation shall be provided to EPA if produced by software. - The median (chip wide, probe level) raw expression level and the median absolute deviation in raw expression level shall not differ more than 3 fold between any two hybridizations in an experiment. Deliverable Contractor shall provide to EPA, in the form of CD or DVD, as soon as raw data is available for each batch set of samples sent by EPA: - RNA QC data and images - scanner settings and image quantitation parameters used throughout experiments - .TIFF files of array images - spot file information with detailed annotation - .xls files of the raw image data Data from any specified replicate hybridizations shall be merged and reported as a single data file in addition to data from each individual experiment. Contractor shall provide cost estimates for 300 samples per array and per array services which would include: - total RNA QC - delivery of RNA QC report - cRNA preparation, labeling, and QC - cRNA hybridization to Agilent zebrafish array - scanning of hybridized microarray - data extraction and QC - delivery of raw data. PERFORMANCE PERIOD: 2 YEARS FROM DATE OF AWARD NOTE: THIS NOTICE WAS NOT POSTED TO WWW.FEDBIZOPPS.GOV ON THE DATE INDICATED IN THE NOTICE ITSELF (10-MAY-2005); HOWEVER, IT DID APPEAR IN THE FEDBIZOPPS FTP FEED ON THIS DATE. PLEASE CONTACT fbo.support@gsa.gov REGARDING THIS ISSUE.
- Web Link
-
The Environmental Protection Agency
(http://www.eps.gov/spg/EPA/OAM/CMD/PR-NC-05-10395/listing.html)
- Record
- SN00804279-F 20050512/050510212554 (fbodaily.com)
- Source
-
FedBizOpps.gov Link to This Notice
(may not be valid after Archive Date)
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