title: UGI Human 12800 V1.0
status: Public on Jan 24 2007
contact.city: Shanghai
contact.first: xuping
contact.last: fu
contact.email: fuxuping@hotmail.com
contact.country: China
contact.institute: Institute of Genetics, Fudan University
contact.phone: 086-021-35013994
organism: Homo sapiens
manufacture_protocol: The construction of the microarrays used in this study was carried out following Brown’s method (available at http://cmgm.stanford.edu/pbrown/protocols/index.html).We fabricated a “genome-wide” cDNA microarray consisting of 12800 sequences including full-length and partial cDNAs representing novel, known, and control genes provided by United Gene Holdings Group Co., Ltd. Furthermore, 12262 clones were obtained from large-scale full-length cloning efforts carried out by the company. 10046 clones were identified with high similarity to the sequences in Unigene database of NCBI by performing Blast (http://www.ncbi.nlm.nih.gov/), while others were novel genes, each of which was given an unique code. The cDNA inserts were amplified by polymerase chain reaction (PCR) with universal primers to plasmid vector sequences and then purified. All the PCR products were examined by agarose gel electrophoresis to ensure the quality and identity of the amplified clones as expected. Then the amplified PCR products were dissolved in a buffer containing 3ХSSC solution. The solution with amplified PCR products were spotted onto silylated slides(CEL Associates, Houston, TX) using a Cartesian PixSys 7500 motion control robot (Cartesian Technologies, Irvine, CA) fitted with ChipMaker Micro-Spotting Technology (TeleChem International, Sunnyvale, CA). Glass slides with spotted cDNA were then hydrated for 2 hr in 70% humidity, dried for 0.5 hr at room temperature, and UV cross-linked (65mj/cm). They were further processed at room temperature by soaking in 0.2% sodium dodecyl sulfate (SDS) for 10 min, distilled H2O for 10 min, and 0.2% sodium borohydride (NaBH4) for 10 min. The slides were dried again and ready for use. The fluorescent cDNA probes were prepared through reverse transcription of the isolated mRNAs and then purified according to the methods of Schena et al.. The noncancerous samples were labeled with Cy3-dUTP. The cancerous samples were labeled with Cy5-dUTP. The two color probes were then mixed, precipitated with ethanol, and dissolved in 20μl of hybridization solution [5ХSSC (0.75 M NaCl and 0.075 M sodium citrate), 0.4% SDS, 50% formamide, and 5ХDenhardt’s Solution (0.1% Ficoll, 0.1% polyvinylpyrrolidone, and 0.1% BSA)]. Resultant cDNA was purified and hybridized to the microarray provided by United Gene Holdings Group Co., Ltd. Microarrays were pre-hybridized with hybridization solution containing 0.5 mg/ml denatured salmon sperm DNA at 42℃ for 6 hr. Fluorescent probe mixtures were denatured at 95℃ for 5 min, and the denatured probe mixtures were applied onto the pre-hybridized chip. And then put a cover glass on it. Chips were hybridized at 42℃ for 15–17 hr. The hybridized chips were then washed at 60℃ for 10 min each in solutions of 2ХSSC and 0.2% SDS, 0.1ХSSC and 0.2% SDS, and 0.1ХSSC, and then dried at room temperature.Every slide was scanned by ScanArray 4000B scanner (General Scanning, Watertown, MA). The fluorescent images of Cy5 and Cy3 were scanned separately and saved for further image analysis. The acquired images were analyzed with GenePix Pro 3.0 software (Axon Instruments, Inc., Union City, CA).