title: Singapore Grouper Iridovirus 0.5K genomic DNA microarray
status: Public on Jan 28 2008
contact.city: Guangzhou
contact.first: yong
contact.last: teng
contact.email: gzyteng@yahoo.com.cn
contact.address: 135 West Xingang Road
contact.country: China
contact.institute: Sun yet-san university
contact.phone: +86-20-84115887
organism: Iridovirus
manufacture_protocol: A SGIV cDNA microarray was constructed following a PCR product-based microarray method. In brief, an in-house high-throughput computer algorithm based on Linux operating system and Phython programming language was employed to design PCR primers for 162 open reading frames (ORFs) of SGIV genome. The fundamental rules of our computer algorithm include that all the primers should have a close annealing temperature with PCR products length about 150-1100 bp and amplicons should have a minimum sequence similarity. The PCR amplifications were performed in 100 µl reaction volume and PCR successes were confirmed by agarose gel electrophoresis. The confirmed PCR products were precipitated with isopropanol and redissolved in 50% dimethyl sulphoxide (DMSO). Two house-keeping genes: Piscine 18S ribosomal RNA (18S rRNA) gene and ß-actin gene from host cells were also amplified to act as positive controls. PCR products (in 50% DMSO, 20 µM) were spotted with SmartArrayTM microarrayer (CapitalBio Corp., Beijing, China). Each gene was printed on amino silaned slides in triplicate to facilitate subsequent statistical analysis. After printing, the slides were baked at 80°C for 1h and stored dry at room temperature till use. Prior to hybridization, the slides were rehydrated over 65°C water for 10 sec. UV cross-linked at 250 mJ/cm2. The unimmobilized DNAs were washed off with 0.5% SDS for 15 min at 42°C and SDS were removed by dipping the slides in anhydrous ethanol for 30 sec. The slides were spin-dried at 1,000 rpm for 2 min.