Shrimp cDNA microarray

Brief protocol

Total RNA extraction and cDNA labeling with Cy3/Cy5

Total RNAs are extracted from the target tissues by using RNeasy Midi Kit (Qiagen) according to the manufacturer’s protocol. Total RNAs of 10 µg are then treated with TURBO DNA-free Kit (Ambion). After adding 1µg poly (dT) primer and 20X low T-dNTP (10mM A, G and CTP, 4mM dTTP), total RNAs are heated at 65℃ for 5 min to denature the secondary structures. Then, Superscript III (Invitrogen) and 1mM Cy3/Cy5-dUTP are added, and reverse transcription proceeds by incubating the reaction at 25℃ for 5 min, 50℃ for 1 h and then 70℃ for 15 min. After reverse transcription, total RNAs are hydrolyzed with 45mM EDTA, 0.2M NaOH at 65℃ for 1hr, and then neutralized with 0.3M Tris-HCl, pH7.6. The unincorporated Cy-dyes are removed by PCR Purification Kit (Qiagen) according to the manufacturer’s protocol, and then the labeled cDNA are concentrated to less than 18 l by using YM-30 microcon (Millipore).

Microarray Hybridization

The slide is pre-hybridized in pre-hybridization buffer (5 X SSC, 0.1% SDS and 1% BSA) for 1 h in a 42℃ water bath. Then, the slide is washed by dunking in MQ water for 2 min, followed by dunking in isopropanol for 2 min, and dried by centrifugation at 500 rpm for 2 min. Before hybridization, the Cy-dye labeled cDNAs are mixed with 1µg Cot-1 DNA and 1 µg poly-A (30 mer), and then heated at 95℃ for 2 min. The denatured cDNAs are mixed with 1 × vol. of hybridization buffer (50% formamide, 10× SSC and 5% SDS) and loaded on the slide. The slide is then covered by a coverslip, put in a hybridization chamber, and then hybridized in a 42℃ water bath for 16 to 24 h. After hybridization, the slide is then washed serially in 2× SSC/0.1% SDS for 10 min, 1× SSC/0.1% SDS for 10 min, 0.2× SSC for10 min and 0.05× SSC for 2 min. Finally, the slide is dried by centrifugation at 500 rpm for 2 min, and is then ready for scanning.

Microarray Analysis

The slides are scanned on GenePix Personal 4100A (Axon Instrument) to measure the fluorescence intensities of Cy3/Cy5 signaling, and are quantified by the software GenePix Pro 6.0 (Axon Instrument). GeneSpring 7.3.1 (Aligent) is used to analyze the preliminary raw data.

References

1. Leu JH, Chang CC, Wu JL, Hsu CW, Hirono I, Aoki T, Juan HF, Lo CF, Kou GH, Huang HC. (2007) Comparative analysis of differentially expressed genes in normal and white spot syndrome virus infected Penaeus monodon. BMC Genomics 8: 120.
2. Tassanakajon A, Klinbunga S, Paunglarp N, Rimphanitchayakit V, Udomkit A, Jitrapakdee S, Sritunyalucksana K, Phongdara A, Pongsomboon S, Supungul P, Tang S, Kuphanumart K, Pichyangkura R, Lursinsap C. (2006) Penaeus monodon gene discovery project: the generation of an EST collection and establishment of a database. Gene 384: 104-12.