Resonance light-scattering particles for ultra-sensitive detection of nucleic acids on microarrays

Abstract

Poster abstracts 67 phases. The first phase is to classify the cDNAs and the second is to complete fulllength 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', which allows the reverse transcriptase reaction at higher (60 C) temperatures. We have constructed over 80 libraries from embryonic tissues of different developmental stages and adult tissues 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 tools developed in-house: a high-throughput plasmid preparation system and the RISA 384 capillary sequencer. Most of the sequences were performed from the 3´ end to select individual cDNAs. We have selected more than 30,000 different cDNAs. Using these sets of RIKEN full-length cDNA, we have established gene expression microarrays containing a 20 K set of RIKEN full-length cDNA unique mouse genes (http://genome.rtc.riken.go.jp). This set has been used to profile expression patterns of various adult and embryonic tissues. Target DNAs were PCR amplified and printed on Poly-L-lysine coated glass slides. Target DNAs were blocked by excess amounts of Cot1DNA. Probes were labelled by two-colour 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 reproducible results were extracted and clustered. We will present a large set of data that show the spatial and temporal expression patterns of mice. These mouse full-length 20 K cDNA microarrays are widely applicable to analyse the global expression profiling of normal and diseased status of mice.