Development of a cDNA array for chicken gene expression analysis
© Burnside et al; licensee BioMed Central Ltd. 2005
Received: 29 September 2004
Accepted: 04 February 2005
Published: 04 February 2005
The application of microarray technology to functional genomic analysis in the chicken has been limited by the lack of arrays containing large numbers of genes.
We have produced cDNA arrays using chicken EST collections generated by BBSRC, University of Delaware and the Fred Hutchinson Cancer Research Center. From a total of 363,838 chicken ESTs representing 24 different adult or embryonic tissues, a set of 11,447 non-redundant ESTs were selected and added to an existing collection of clones (4,162) from immune tissues and a chicken bursal cell line (DT40). Quality control analysis indicates there are 13,007 useable features on the array, including 160 control spots. The array provides broad coverage of mRNAs expressed in many tissues; in addition, clones with expression unique to various tissues can be detected.
A chicken multi-tissue cDNA microarray with 13,007 features is now available to academic researchers from firstname.lastname@example.org. Sequence information for all features on the array is in GenBank, and clones can be readily obtained. Targeted users include researchers in comparative and developmental biology, immunology, vaccine and agricultural technology. These arrays will be an important resource for the entire research community using the chicken as a model.
The chicken is an important experimental model for evolutionary and developmental biologists, immunologists, cell biologists, geneticists, as well as being an important agricultural commodity. The recent release of a draft of the chicken genome sequence, as well as the development of a large (531,351) collection of expressed sequence tags (ESTs) has dramatically changed the landscape for biologists wishing to use genomic tools to study the chicken. DNA microarrays are well accepted as an essential part of functional genomics. Several small chicken cDNA arrays have been fabricated and used in studies focused on the chicken immune system [1–4]. To enhance the utilization of existing resources and further develop the chicken as a model organism, a consortium was formed to produce microarrays using clones from the Biotechnology and Biological Sciences Research Council (BBSRC), University of Delaware (UD) and Fred Hutchinson Cancer Research Center (FHCRC). The BBSRC chicken cDNA project generated a large (>300,000) collection of ESTs that represents a wide range of adult and embryonic tissues . The UD Chick EST project has focused on tissues important in agricultural production, with a heavy emphasis on the immune system . The FHCRC EST collection was generated from DT40 cells (a transformed bursal cell line) [1, 2], along with clones from the bursal EST project [7, 8] and the UD activated T cell library . By combining resources and clones from these projects, we have established a collection that encompasses a variety of tissues, and generated microarrays with 13,007 usable features. This paper describes the array with respect to clone selection and quality control parameters.
Results and discussion
Selection of clones for the array
A list of the clones can be accessed on-line . The clones represented in the list total 15,769. PCR product quality was assessed using gel electrophoresis and the results were meticulously scored and recorded. After identifying poor quality PCR products (e.g., no detectable product, detection of multiple products), the number of useable features totals approximately 13,000, including control features. The annotation file contains accession numbers, source clone name, and source assigned annotation or Blast derived annotation. In addition the EST identification assigned by The Institute for Genome Research (TIGR) and found in TIGR's Gallus gallus Gene Index (GgGI)  is provided, as is the identifier for TIGR's consensus (TC) sequence and TIGR annotation. An analysis of the TC identifiers for clones on the array revealed that 1,184 mRNAs are represented by more than one clone. This is due to clones in non-overlapping contigs and some redundancy in the original immune collection. A more detailed annotation file, as well as a database for array data is under development and will be accessible on line .
Clone selection and array fabrication predated the sequencing of the chicken genome. An analysis of the sequence of the clones on the array indicates that 10,168 of the 21,447 predicted or annotated chicken genes in the GenBank chicken Unigene collection are present on the array. The remaining clones match cDNAs not yet included in Unigene, or other portions of the chicken genome, or are redundant.
Clones are available from their original source: the BBSRC collection, distributed by the MRC gene service ; the DKFZ collection at Heinrich-Pette-Institute maintained by Dr. Jean-Marie Buerstedde ; the DT40 collection at Fred Hutchinson Cancer Research Center, maintained by Dr. Paul Neiman ; the T-cell and lymphoid libraries, maintained by Dr. Joan Burnside of the Delaware Biotechnology Institute .
Chicken 13K array performance
Signal-to-noise, specificity and sensitivity
Chicken 13K cDNA Microarray Performance Metrics
Label / Sample
Mean BG Signal
Spot-Level S/N >3
Mean Spot-Level S/N
Cy3 / Fibroblast
118 ± 6
Cy5 / Brain
48 ± 3
In a separate experiment, T7 amplified, random-primer labeled RNA was compared with random-primer labeled poly A RNA (from the same preparation). Figure 8C shows a fair concordance with about 80% of the same spots showing hybridization with each sample. However, this comparison reveals that amplification loses some signals detected with mRNA but picks up others, presumably from low abundance messages which amplify better (with respect to the cDNA sequences on the chip) than average. In another experiment (not shown) repeat amplifications of the same RNA prep give satisfactorily consistent results (correlation coefficient >0.9). These results emphasize that it is important to use the same method of RNA preparation and labeling to obtain reliable comparisons.
An international consortium of researchers interested in using the chicken as both a model biological system and as an important agricultural commodity have consolidated resources to produce a microarray containing 13,000 features representing approximately 12,000 different mRNAs. These are now available to academic researchers through email@example.com. This array overlaps previous chicken immunology arrays and extends the coverage to 24 different tissues or cell types. In conjunction with the recent release of the chicken genome sequence, this tool will have wide application to studies in developmental biology, immunology, vaccine application, as well as identification of well-characterized complex traits. The availability of genomics tools will enhance the further development of the chicken as a powerful biological model.
Materials and methods
Libraries and array construction
BBSRC and UD clones were shipped to the FHCRC core genomics lab. Information on the libraries joined to produce this collection is available at individual web sites and previous publications [1, 6, 11, 15]. Microarrays were constructed using modified protocols of those discussed by De Risi et al. . Individual PCR products were verified as unique via gel electrophoresis and purified using the Millipore Multiscreen-PCR filtration system. Purified PCR products were mechanically "spotted" in 3X SSC (1X = 150 mM sodium chloride, 15 mM sodium citrate, pH 7.0) onto poly-lysine coated microscope slides using a GeneMachines OmniGrid high-precision robotic gridder (Genomics Solutions, Ann Arbor, MI). The array layout consists of 32 blocks in a 4 × 8 configuration and each PCR product is represented once on the array. In addition, each array sub-grid (i.e., "block") contains spots representing 4 different Arabidopsis genes (negative controls) and 1 spot consisting of sheared chicken (white leghorn) genomic DNA.
A GenePix scanner-compatible file (chicken 13k_v1.0.gal) is available on line . For other scanners, this file can be opened in a text editor and used to construct a similar file that meets other image analysis software's format specifications.
RNA preparation, labeling and hybridization
Total RNA was prepared using Qiagen (Chatsworth, CA) RNeasy kits and amplified using a linear T7 promoter-based mRNA amplification method incorporating amino -allyl dUTP followed by random primer labeling with Cy™3 or Cy™ 5 (Amplification and labeling kits are available from Ambion, Inc., Austin, TX).
For hybridization, 10%, sodium dodecyl sulfate (SDS), 0.6 μl was added to the labeled RNA and heated at 99 C for 2 min. RNA was then centrifuged at 14,000 rpm for 3 min, and the sample cooled to room temperature. After placing an array slide in a hybridization chamber, 10 μl 3X SSC was added to the slide, away from the spotted area. RNA sample was then added to the array area and the cover slip promptly positioned over the array. The sealed hybridization chamber was incubated in a water bath at 63 C for 16 h. The slide was then washed for 2 min in a standard slide washing container, first in 1X SSC/0.03% SDS, then in 1X SSC, followed by a 20 min wash with agitation (60 rpm) in 0.2X SSC and a 10 min wash with agitation in 0.05X SSC. The slide was protected from light during the prolonged washes. The slide was then centrifuged (500 rpm × 5 min) to dry. Fluorescent array images were collected for both Cy3™ and Cy5™ using a GenePix 4000A fluorescent scanner (Axon Instruments, Inc., Foster City, CA) and image intensity data was extracted and analyzed using GenePix Pro 3.0 microarray analysis software.
JB and PN generated UD and FHCRC clones, respectively. DB provided the BBSRC clones. JB and JT performed the analysis for clone selection. JD and RB fabricated the microarrays. PN, JD, RB performed the analysis and validation of the microarray. MA generated the annotation file.
This work was supported in part by FHCRC Pilot Project Funds and NIH grant R01 CA20068 to PN, the UD Chick EST project, the US Poultry Genome project (Hans Cheng and Jerry Dodgson), USDA-NRI grant 00-35205-9407 to JB and Robin Morgan.
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