title: UKM Lates calcarifer liver and spleen cDNA array
status: Public on Aug 07 2012
contact.city: Bangi
contact.first: Abdul Munir
contact.last: Abdul Murad
contact.email: munir8488@gmail.com, munir@pkrisc.cc.ukm.my
contact.state: Selangor
contact.address: Jalan Universiti
contact.department: Faculty of Science & Technology
contact.country: Malaysia
contact.institute: Universiti Kebangsaan Malaysia
organism: Lates calcarifer
manufacture_protocol: Using the GeneTac™G3 (Genomic Solutions™, USA) arrayer, we fabricated the in-house Asian seabass (Lates calacrifer) cDNA microarray slide, comprising 1920 probes sourced from in-house Lates calcarifer spleen and liver cDNA libraries. Putative functions encoded by these probes include immunity, stress response, metabolism, transport, transcription and translation. Negative controls, such as oligonucleotide (A)50 and several Saccharomyces cerevisiae and Escherichia coli genes, as well as positive controls, such as a serial dilution of L. calcarifer genomic DNA and pooled cDNA, were also included. PCR amplification of the clones was carried out using a pair of M13 universal primers (Forward 5’-GTAAAACGACGGCCAG-3’, Reverse 5’-GTCATAGCTGTTTCCTG-3’) under the following conditions: initial denaturation at 95oC for 2 min, followed by 30 cycles of 95oC for 30 sec, 52 oC for 30 sec and 72 oC for 2 min, and a final extension step at 72 oC for 10 min. All the amplicons were checked with agarose gel electrophoresis and subsequently purified using Montage® PCRµ96 Plate (MilliPore, USA). The amplicon was resuspended in DMSO with the final concentration of probes ranging from 150-250 μg/mL. The probes were spotted twice onto aminosilane-coated glass slides (Corning, USA). The array consists of spots arranged in 4 x 8 grids. There are 32 grids in 4 columns (meta-columns) and 8 rows (meta-rows). Each of the grids contained 11 x 11 spots generating 121 spots per grid. Each fabricated slide contained a total of 3872 spots including 32 GeneTac™G3 arrayer prefix empty spots. Each spotted slide was left overnight in the arrayer. Subsequently, the slide was crosslinked at 300 mJ of ultraviolet (UV) irradiation with HoeferTM UVC 500 Crosslinker (Amersham Biosciences, UK) and stored in humidity and light-controlled conditions until use. Quality of the slide produced was assessed by a hybridization check with a random nanomer from the SpotCheckTM  Slide QC kit (Genetix, USA).