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KMID : 0358920050320010075
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Journal of the Korean Academy of Pedodontics
2005 Volume.32 No. 1 p.75 ~ p.88
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ANALYSIS OF DIFFERENTIAL GENE EXPRESSION IN NORMAL, CYST AND AMELOBLASTOMA CELLS
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Yang Cheol-Hee
Kim Jae-Gon
Baik Byeong-Ju
Yang Yeon-Mi
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Abstract
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Ameloblastoma is the most commonly occurring odontogenic tumor in oral cavity. Although most are benign epithelial neoplasm, they are generally considered to be locally aggressive and destructive, exhibiting a high rate of recurrence. The biological behavior of this neoplasm is a slowly growing, locally invasive tumor without metastasis, therefore malignant neoplasm, changed its histological appearance to carcinoma or showed distant metastasis, is only defined clinically.
In this study, we identified the differentially expressed genes(DEGs) in stages under benign or malignant ameloblastoma compared with normal patient using ordered differential display(ODD) reverse transcription(RT)-PCR and GeneFishing™ technology. ODD RT-PCR is rather effective when the investigation of samples containing very small amounts of total RNA must be accomplished. ODD RT-PCR used the means of amplification with anchored T-primer and adaptor specific primer, bearing definite two bases at their 3’ ends and so this method could display differential 3’ -expressed sequence taqs(ESTs) patterns without using full-length cDNAs. Compared with standard differential display, ODD RT-PCR is more simple and have enough sensitivity to search for molecular markers by comparing gene expression profiles. However, this method required much effort and skill to perform.
GeneFishing™ modified from DD-PCR is an improved method for detecting differentially expressed genes in two or more related samples. This two step RT-PCR method uses a constant reverse primer(anchor ACP-T) to prime the RT reaction and arbitrary primer pairs(annealing control primers, ACPs) during PCR. Because of high annealing specificity of ACPs than ODD RT-PCR, the application of GeneFishing™ to DEG discovery generates reproducible, authentic, and long(100bp to 2kb) PCR products that are detectable on agarose gels.
Consequently, various DEGs observed differential expression levels on agarose gels were isolated from normal, benign, and malignant tissues using these methods. The expression patterns of the some isolated DEGs through ODD RT-PCR and GeneFishing™ were confirmed by Northern blot analysis and RT-PCR. The results showed that these identified DEGs were implicated in ameloblastoma neoplasm processes. Therefore, the identified DEGs will be further studied in order to be applied in candidate selection for marker as an early diagnosis during ameloblastoma neoplasm processes.
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KEYWORD
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Ameloblastoma, ODD RT-PCR, GeneFishing™
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