Tucson, Arizona
University of Arizona
Maize cDNA microarrays --Elumalai, R, Gchachu, M, Pierson, E, Chandler, V, Galbraith, DW Microarray technology has revolutionized gene expression studies by providing a powerful approach to examine gene expression patterns of tens of thousands of genes at a time [M. Schena et al. 1995. Quantitative monitoring of gene-expression patterns with a complementary-DNA microarray. Science 270:467-470; M.K. Deyholos and D.W. Galbraith 2001. High-density DNA microarrays for gene expression analysis. Cytometry 43:229-238]. One component of the NSF-funded Maize Gene Discovery project led by Virginia Walbot at Stanford University (DBI- 9872657) is to produce cDNA microarrays for maize genes and distribute these to the research community at a reasonable cost. In this report (December 2001), we summarize the status of the maize microarrays. Below we give an overview on the production and summarize the content of each array produced to date. For the latest information, please see the project website at www.zmdb.iastate.edu.

At Stanford University over 100,000 ESTs have been sequenced from cDNA libraries constructed from different tissue types. The Arizona team receives a copy of the sequenced EST libraries for microarray production. The EST sequences are amplified by polymerase chain reaction (PCR) using universal primers in 96 well format. The PCR products are analyzed by gel electrophoresis for the quality of amplification and the approximate size of the PCR products are estimated and cataloged. The PCR products are then purified using the Millipore DNA purification system, dried in a Speed-vac, and re-suspended in buffer for microarray printing. The microarrays are printed onto amino-silane coated microscopic slides using a GeneMachines Omnigridder. For quality control, exemplar slides from each printing are subjected to several procedures prior to distribution. These include staining with DNA intercalating dye propidium iodide to determine the degree of uniformity of spots, and hybridization using targets prepared from mRNA isolated from the same type of tissue that was used for the original cDNA library construction. Information about every EST printed on each microarray is provided on our project web site, http://gremlin3.zool.iastate.edu/zmdb/microarray/ . Included in this information is the size of the PCR amplification product, the sequence annotation, the location of each EST on the microarray and images providing hybridization examples. We also provide details of the primers used for the amplification of each library, and our methods for PCR amplification, slide production, RNA isolation, mRNA purification, mRNA labeling and slide hybridization. We are also available to answer any questions and provide help as needed. Contact information can be found at the website. To date we have distributed 600 slides to 55 laboratories and hosted 15 visitors to the laboratory.

605 Endosperm microarrays. The 605 microarray comprises cDNAs from developing endosperm (14 days post pollination). It contains 8064 ESTs, representing approximately 2000-3000 unique genes. Each EST is spotted twice on the microarray array either in duplicate array format or in duplicate spot format. The 605 microarrays were validated by hybridizing with fluorescent labeled mRNAs isolated from 14 day-old immature endosperm. The reproducibility was high, within a slide the R values ranged from 0.92 to 0.98 for the individual channel intensity values and between the slides R values ranged from 0.77 to 0.95. Details are provided on the web site and in Fernandes et al., 2002, Plant Physiology, in press.

606 Immature ear microarrays. The 606 microarrays comprise cDNAs from developing ear (0.5-2 cm long), and contain 5065 ESTs, representing an estimated 2000 genes. Cherry picking was implemented to reduce the redundancy of cDNAs on this microarray to a maximum of 2 3 X; also, ESTs that did not produce good quality sequence were not printed. In the 606 microarrays, each EST is printed in triplicate with the three spots immediately adjacent to each other. The 606 microarrays were validated by hybridizing with fluorescent targets produced from mRNA of 2-3 cM long developing ears. The reproducibility was high, within a slide the R values ranged from 0.92 to 0.98 for the individual channel intensity values and between the slides R values ranged from 0.61 to 0.92. Details are provided on the web site and in Fernandes et al., 2002, Plant Physiology, in press.

614 Root microarrays. The 614 microarrays comprise cDNAs from developing roots (3-4 days old), and contain 5065 ESTs, representing 2000 2500 unique genes. As for the 606 microarrays, we reduced the redundancy of abundant ESTs to 2 3 X, removed those ESTs that did not produce good quality sequence, and printed three adjacent spots for each EST. The 614 microarrays were validated by hybridizing with fluorescent target prepared from mRNA of 4 day-old developing roots. The reproducibility was high, within a slide the R values ranged from 0.91 to 0.96 for the individual channel intensity values and between the slides R values ranged from 0.78 to 0.96.

486 Immature leaf microarrays. The 486 microarrays comprise cDNAs from developing leaf primordia (P4/6-P10/11 leaf primordial stage), and contain 4454 ESTs, representing an estimated 2000 unique genes. As for the 606 and 614 arrays, we have reduced the redundancy of abundant ESTs to 2 3 X, removed those ESTs that did not produce good quality sequence, and printed three adjacent spots for each EST. The 486 microarrays were tested by hybridizing with fluorescent target prepared from mRNA of the P4/6-P10/11 leaf primordial stage. The reproducibility within a slide was high with a R value of 0.94 for the individual channel intensity values.

Unigene 1-1-01 microarrays. The unigene 1-1-01 microarrays are the first component of our Unigene microarray set, which is designed to contain unique genes without redundancy. The unigene 1-1-01 microarrays each contain 5280 unique genes from 3 different cDNA libraries (707&945; mixed adult tissues, 687; developing embryo and 603; salt stressed root). The unigene 1-1-01 array was printed in a triplicate spot format. The Unigene 1-1-01 array has been validated by hybridization with cy5 labeled target from mRNA of husk tissue. The reproducibility within a slide was high with a R value of 0.96 for the individual channel intensity values.

Summary. The microarrays described above are available by ordering from the project website. Our plans for the coming year are to print the rest of the consolidated Unigene library, which will contain a total of 15000 estimated unique genes. As the additional slides become available, this will be announced on the project website.
 
 


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