Cancer Genetics and Cytogenetics 152 (2004) 124–128 Short communication Amplification of MGC2177, PLAG1, PSMC6P , and LYN in a malignant mixed tumor of salivary gland detected by cDNA microarray with tyramide signal amplification Yvonne T.M. Tsang, Yi-Mieng Chang, Xinyan Lu, Pulivarthi H. Rao, Ching C. Lau, Kwong-Kwok Wong* Texas Children’s Cancer Center, Cancer Genomics Group, MC3-3320, Department of Pediatrics, Baylor College of Medicine, 6621 Fannin Street, Houston, TX 77030 Received 3 October 2003; received in revised form 1 December 2003; accepted 2 December 2003 Abstract Gene amplifications have been observed in many different tumor cells, and many of these changes are related to tumor pathogenesis. Comparative genomic hybridization (CGH) using metaphase chromosomes can detect changes in chromosome copy number with a resolution of 10–20 Mb. Current advances in CGH analysis in a microarray format allow us to refine such changes down to the gene level. We applied microarray technology to detect novel gene amplification in a malignant mixed tumor of salivary gland. Besides detecting previously known gene amplifications (MDM2 and MYC), we identified four other highly amplified genes located at 8q11.2~q13: MGC2177, PLAG1, PSMC6P , and LYN. The amplification was further validated with real-time quantitative polymerase chain reaction.  2004 Elsevier Inc. All rights reserved. 1. Introduction The gain, loss, or amplification of chromosome regions is common among tumor cells. Genome-wide scanning of chromosome copy numbers with comparative genomic hy- bridization (CGH) on metaphase chromosomes is a powerful technique that has revealed many chromosomal aberrations in tumor cells, but with a limited resolution of 10–20 Mb [1]. To improve the resolution of conventional chromosome CGH, array CGH has been applied with use of cloned geno- mic DNA such as bacterial artificial chromosomes (BACs) [2–4], P1 clones [5,6], and cDNA clones [7,8]. Using chro- mosome CGH, we earlier identified a few chromosome re- gions with amplification in a malignant mixed tumor of salivary gland [9]. To further investigate the molecular struc- ture of the amplicons in this salivary gland tumor, we applied cDNA microarrays for CGH analysis, a technique that has potentially much higher resolution than chromosome CGH. * Corresponding author. Tel.: (832) 824-4373; fax: (832) 825-4038. E-mail address: kkwong@bcm.tmc.edu (K.-K. Wong). 0165-4608/04/$ – see front matter  2004 Elsevier Inc. All rights reserved. doi:10.1016/j.cancergencyto.2003.12.001 2. Materials and methods 2.1. Differential random primer labeling Genomic DNA (50 ng) derived from a normal placenta and from malignant mixed tumor cells of salivary gland was digested with restriction enzyme EcoRI overnight in a volume of 20 μL of 1× universal restriction buffer (Stratagene, La Jolla, CA) at 37°C. After digestion, the restriction enzyme activity was inactivated by heating at 70°C for 20 minutes. Subsequently, 20 μL of 2.5× Random Primer solution from the BioPrime DNA labeling system (Invitrogen, Carlsbad, CA) was added to the 20 μL digested genomic DNA to obtain a final volume of 40 μL. The mixture was heated for 5 minutes in boiling water and then was placed on ice. Two sets of DNA, one from tumor cells and the other a refer- ence genomic DNA, were differentially labeled with biotin and fluorescein using random primers. Next, 5 μL biotin- 11-dCTP (1 mmol/L; PerkinElmer Life Sciences, Boston, MA) and 3 μL unlabeled dNTPs (1.7 mmol/L dCTP, 3.3 mmol/L dGTP, 3.3 mmol/L dATP, and 3.3 mmol/L dTTP) was added to the 40 μL tumor DNA reaction mix; 5 μL of 1 mmol/L fluorescein-12-dCTP (PerkinElmer Life Sciences) was added to the reference DNA. Finally, 2 μL of Klenow fragment (Invitrogen) was added to both reaction mixes to