|Function and cDNA Resources Section
DOE Human Genome Program Contractor-Grantee
100. Tissue Gene Expression Profiling Using RIKEN Full-Length Mouse 20K cDNA Microarray
Yasushi Okazaki1,2, Rika Miki1,2,3, Yosuke Mizuno1,2,3, Yasuhiro Tomaru1, Kouji Kadota1,2,4, Piero Carninci1, Kazuhiro Shibata1,2, Masayoshi Itoh1,2, Yasuhiro Ozawa1, Jun Kawai1,2, Hideaki Konno1,2, Yoshifumi Fukunishi1,2, Toshinori Kusumi1, Hitoshi Goto1,5, Hiroyuki Nitanda1,5, Yohei Hamaguchi1,6, Itaru Nishiduka1,6, Masami Muramatsu1,2, Atsushi Yoshiki7, Moriaki Kusakabe7, Joseph Derisi8, Vishy Iyer9, Michael Eisen9, Patric O. Brown9, and Yoshihide Hayashizaki1,2,3
1Laboratory for Genome Exploration Research Project, Genomic Sciences Center (GSC) and Genome Science Laboratory, Tsukuba Life Science Center, The Institute of Physical and Chemical Research (RIKEN), Koyadai, Japan; 2CREST, Japan Science and Technology Corporation (JST); 3Tsukuba University; 4University of Tokyo; 5Tohoku University, Sendai, Japan; 6Yokohama City University; 7Experimental Animal Research Division, Tsukuba Life Science Center, The Institute of Physical and Chemical Research (RIKEN), Koyadai, Japan; 8University of California; and 9Stanford University, Stanford, CA
The target of the Genome Science Laboratory of RIKEN is to clone and sequence the largest number possible of full-length mouse cDNAs and then to sequence these cDNAs in two phases. The first phase is to classify the cDNAs and the second is to complete full-length sequencing and functional annotations. We have developed two original methods to construct full-length cDNAs efficiently: "cap-trapper" which preferentially recognizes the Cap site of mRNA and the "trehalose-thermoactivated reverse transcriptase (RT)" which allows the RT reaction at higher (60 C) temperature. We have constructed over 80 libraries from embryonic tissues of different developmental stages and adult tissues in order to ensure the greatest possible coverage of the expressed mRNA.
More than 200,000 successful sequencing passes have been performed with the use of two in house developed tools; a high-throughput plasmid preparation system and the RISA 384 capillary sequencer. Most of the sequences were performed from 3' end in order to select individual cDNAs. We have selected more than 65,000 different cDNAs.
Using these sets of RIKEN full-length cDNA, we have established Gene Expression Microarrays containing 20 K set of RIKEN full-length cDNA unique mouse genes (http://genome.rtc.riken.go.jp). These set have been used to profile expression patterns of various adult and embryonic tissues. Target DNAs were PCR amplified and printed on the Poly-L-lysine coated slide glasses. Target DNAs were blocked by excess amount of Cot1DNA. Probes were labeled by two-color fluorescent dye using random primer and reverse transcriptase. Normalization has been achieved using a global normalization method. We have also developed a program to filter the noise. The experiment was done twice and the reproducible results were extracted and clustered. We will present a large set of database, which show the spatial and temporal expression patterns of mice. These mouse full-length 20 K cDNA microarrays are widely applicable to analyze the global expression profiling of normal and diseased status of the mice.
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