Sains Malaysiana 38(4)(2009): 609–617

Fabrikasi Slaid Mikroatur cDNA Lates calcarifer

(Fabrication of Lates calcarifer cdnaMicroarray Slide)

Khoo Choon Kiat^{1, 2}, Adura Mohd Adnan^1,2, Kua Beng Chu³ & Abdul Munir Abdul Murad^{1, 2} *,

¹Pusat Pengajian Biosains dan Bioteknologi, Fakulti Sains dan Teknologi

Universiti Kebangsaaan Malaysia, 43600 UKM Bangi, Selangor D.E., Malaysia

²Institut Genom Malaysia

Blok Heliks Emas, Pusat Pintar UKM-MTDC, Universiti Kebangsaaan Malaysia

43600 UKM Bangi, Selangor D.E., Malaysia

³Pusat Penyelidikan Kesihatan Ikan Kebangsaan

Institut Penyelidikan Perikanan

11960 Batu Maung, Pulau Pinang, Malaysia

Diserahkan: 14 Julai 2008 / Diterima: 20 November 2008

ABSTRAK

Teknologi DNA mikroatur merupakan salah satu teknologi penting dalam penyelidikan biologi kini. Teknologi tersebut membolehkan analisis pengekspresan gen pelbagai sistem model pada skala genom dijalankan. Prestasi DNA mikroatur ditentukan oleh parameter seperti kepadatan titik, ciri-ciri titik (morfologi, kepadatan prob dan keamatan isyarat penghibridan), latar belakang, kespesifikan dan kesensitifan. Dengan menggunakan mesin pemegun GeneTac™G3, slaid mikroatur cDNA ikan siakap (Lates calcarifer) yang mengandungi 1920 klon-klon cDNA terpilih daripada perpustakaan cDNA hepar dan limpa yang dipegunkan secara duplikasi telah difabrikasikan. Klon-klon yang dipilih mempunyai fungsi putatif berkaitan dengan keimunan, aruhan tekanan, metabolisme, pengangkutan, transkripsi dan translasi. Kawalan negatif seperti oligonukleotida (A)50 dan beberapa gen daripada Saccharomyces cerevisiae dan Escherichia coli serta kawalan positif seperti beberapa siri pencairan DNA genom dan cDNA L. calcarifer juga dipegunkan ke atas slaid kaca. Diperhatikan bahawa kepekatan prob dalam julat antara 150-250 μg/mL, kesesuaian jenis kawalan positif dan negatif yang digunakan berjaya menghasilkan slaid yang berkualiti. Selain itu, perlakuan pengeringan slaid secara semalaman dan rehidrasi semula slaid menghasilkan morfologi titik DNA yang sekata dan membulat. Kawalan kualiti ke atas slaid yang terhasil membuktikan keberkesanan slaid yang difabrikasi untuk kajian respons transkriptom L. calcarifer terhadap jangkitan Cryptocaryon irritans.

Kata kunci: Lates calcarifer; mikroatur DNA; profil transkriptom

ABSTRACT

DNA microarray technology has become an essential part of biological research. It enables the genome-scale analysis of gene expression of various model systems performed. DNA microarray performance is measured by parameters like spot density, spot characteristics (morphology, probe density and hybridised intensity), background, specificity and sensitivity. Using the GeneTac™G3 arrayer, we fabricated the Asian seabass (Lates calacrifer) cDNA microarray, comprising 1920 selected cDNAs clones sourced from spleen and liver cDNA libraries, and spotted in duplicate. Putative functions encoded by these clones 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 cDNA were also spotted onto the glass slide. It was observed that the probes concentration ranging from 150-250 μg/mL and the type of positive and negative control employed produced quality slide. In addition, the overnight drying of the spotted slides and slide rehydrating produced morphologically even and round DNA spots. Slide quality controls employed demonstrated the functionality of the fabricated slides in profiling the L. calcarifer transcriptome response toward Cryptocaryon irritans infection.

Keywords: DNA microarray; Lates calcarifer; transcriptome profiling

RUJUKAN

Albrecht, V., Chevallier, A., Magnone, V., Barbry, P., Vandenbos, F., Bongain, A., Lefebvre, J-C. & Giordanengo, V. 2006. Easy and fast detection and genotyping of high-risk human papillomavirus by dedicated DNA microarrays. Journal of Virological Methods 137(2): 236-244.

Bai, Q., McGillivray, C., da Costa, N., Dornan, S., Evans, G., Stear, M.J. & Chang, K-C. 2003. Development of a porcine skeletal muscle cDNA microarray: analysis of differential transcript expression in phenotypically distinct muscles. BMC Genomics 4(8). http://www.biomedcentral.com/1471-2164/4/8 [30 Januari 2007].

Byon, J.Y., Ohira, T., Hirono, I. & Aoki, T. 2005. Use of a cDNA microarray to study immunity against viral hemorrhagic septicemia (VHS) in Japanese flounder (Paralichthys olivaceus) following DNA vaccination. Fish and Shellfish Immunology 18: 135-147.

Chandrasekharappa, S., Holloway, A., Iyer, V., Monte, D., Murphy, M. & Nowak, N. J. 2003. Generation of probes for spotted microarrays. Dlm. DNA microarrays: A molecular cloning manual, Bowtell, D. & Sambrook, J. (pnyt). hlm. 1-60. New York: Cold Spring Harbor Laboratory Press.

Childs, G., Desiri, J., Harris, T., Holloway, A., Hou, B.H., Massimi, A., Murphy, M. & Somerville, S. 2003. Printing spotted glass microarrays. Dlm. DNA microarrays: A molecular cloning manual, Bowtell, D. & Sambrook, J. (pnyt). hlm. 61-97. New York: Cold Spring Harbor Laboratory Press.

Crowther, D.J. 2002. Application of microarrays in the pharmaceutical industry. Current opinion in Pharmacology 2: 551-554.

Debouck, C. & Goodfellow, P.N. 1999. DNA microarrays in drug discovery and development. Nature Genetics Supplement 21: 48-50.

Duggan, D.J., Bittner, M., Chen, Y., Meltzer, P. & Trent, J.M. 1999. Expression profiling using cDNA microarrays. Nature Genetics Supplement 21: 10-14.

Dufva, M. 2005. Fabrication of high quality microarrays. Biomolecular Engineering 22: 173-184.

Ewart, K.V., Belanger, J.C., williams, J., Karakach, T., Penny, S., Tsoi, S.C.M., Richards, R.C & Douglas, S.E. 2005. Identification of genes differentially expressed in Atlantic salmon (Salmo salar) in response to infection by Aeromonas salmonicida using cDNA microarray technology. Developmental and Comparative Immunology 29(4): 333-347.

Gracey, A.Y., Troll, J.V. & Somera, G.N. 2001. Hypoxia induced gene expression profiling in the euryoxic fish Gillichthys mirabilis. Proceedings of National Academic of Science 98: 1993-1998.

Hegde, P., Qi, R., Abernathy, K., Gay, C., Dharap, S., Gaspard, R., Earle-Hughes, J., Snesrud, E., Lee, N. & Quackenbush, J. 2000. A Concise Guide to cDNA Microarray Analysis-II. Biotechniques 29(3): 548-562.

Holloway, A.J., van Laar, R.K., Tothill, R.w. & Bowtell, D.L. 2002. Options available-from start to finish-for obtaining data from DNA microarrays II. Nature Genetics Supplement 32: 481-489.

Howbrook, D.N., van der Valk, A.M., O’Shaughnessy, M.C., Sarker, D.K., Baker, S.C. & Lloyd, A.w. 2003. Developments in microarray technologies. Drug Discovery Today 8: 642-651.

Khoo, C. K. 2007. Analisis transkriptom respons imun Lates calcarifer terhadap jangkitan Cryptocaryon irritans. Tesis Sarjana. Universiti Kebangsaan Malaysia.

Matson, R. S. 2004. Applying Genomic and Proteomic Microarray Technology in Drug Discovery, hlm.93-146. US: CRC.

Meijer, A.H., Verbeek, F.J., Salas-Vidal, E., Corredor-Ad‡mez, M., Bussman, J., van der Sar, A.M., Otto, G.w., Geisler, R. & Spaink, H.P. 2005. Transcriptome profiling of adult zebrafish at the late stage of chronic tuberculosis due to Mycobacterium marinum infection. Molecular Immunology 42(10): 1185-1203.

Nurul Yuziana, M.Y. 2004. Penjanaan dan analisis penanda jujukan terekspres (EST) daripada Lates calcarifer. Tesis Sarjana. Universiti Kebangsaan Malaysia.

Rise, M.L., Jones, S.R.M., Brown, G.D., von Schalburg, K.R., Davidson, W.S. & Koop, B.F. 2004. Microarray analyses identify molecular biomarkers of Atlantic salmon macrophage and hematopoietic kidney response to Piscirickettsia salmonis infection. Physiology Genomics 20: 21-35.

Roberge, C., P‡ez, D.J., Rossignol, O., Guderley, H., Dodson, J. & Bernatchez, L. 2007. Genome-wide survey of the gene expression response to saprolegniasis in Atlantic salmon. Molecular Immunology 44(6): 1374-1383.

Sambrook, J. & Russel, D.w. 2001. Cloning: A Loboratory Manual. Ed. ke-3. New York: Cold Spring Harbor Laboratory Press.

Schena, M. 2002. Microarray Analysis. New Jersey: John Wiley & Son.

Venkatasubbarao, S. 2004. Microarray-status and prospects. Trends in Biotechnology 22(12): 630-637.

Yue, H., Eastman, P.S., wang, B.B., Minor, J., Doctolero, M.H., Nuttall, R.L., Stack, R., Becker, J.w., Montgomery, J.R., Vainer, M. & Johnston, R. 2001. An evaluation of the performance of cDNA microarrays for detecting changes in global mRNA expression. Nucleic Acids research 29:e41. http://nar.oxfordjournals.org/cgi/reprint/29/8/e41 [30 Januari 2007].

*Pengarang untuk surat-menyurat; email: munir@ukm.my