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KMID : 0354720070310050410
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Journal of Korean Diabetes Association
2007 Volume.31 No. 5 p.410 ~ p.420
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Transcription Factor Profile by Degenerate RT-PCR/SSCP - Application in 3T3-L1 Adipocyte Treated with TNF-a-
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Kim Yoo-Lee
Choi Young-Kil
Park Seok-Won
Cho Yong-Wook
Lee Sang-Jong
Kim Soo-Kyung
Chang Seo-Yoon
Lee Sang-Hwa
Kim Yun-Soo
Park Won-Keun
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Abstract
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Background: Several high-throughput gene analysis techniques - differential display PCR, suppression subtraction hybridization (SSH), serial analysis of gene expression (SAGE), and DNA microarray - have permitted transcriptome profiling to understand the molecular pathogenesis of multifactorial diseases. But these techniques are of no great utility regarding feasibility, reproducibility, cost, and the amount of material required for analysis. To establish more practical method for transcription factor transcriptome profiling, we combined degenerate reverse transcriptase-polymerase chain reaction (RT-PCR) and single strand conformational polymorphism (SSCP) technique.
Methods: We categorized 417 human/mouse transcription factor mRNA into 92 small groups according to homology with ClustalW method and established 92 degenerate RT-PCR including common motives of the 92 small groups with the software program of CODEHOP, Primer Premier, Amplify 1.2. Further analysis on the amplified PCR products was performed by SSCP. This system was applied for the evaluation of changes on transcription factor transcriptome of differentiated 3T3-L1 adipocyte treated with TNF-alpha.
Results: 82 groups and 52 groups showed amplification of PCR before and after TNF-alpha treatment respectively and 24 groups showed significant amplification difference after TNF-alpha treatment. After TNF-alpha treatment for 48 hours, mRNA expressions of group 7, 30, and 33 which include adipocyte related transcription factors such as CEBP-alpha, RXR-alpha, PPAR-gamma were downregulated and mRNA expression of group 8 including preadipocyte abundant CEBP-beta was upregulated. These results are largely concordant with the results analyzed by oligonucleotide microarray. Randomly selected single PCR bands of group 28 and 75 on agarose electrophoresis displayed additional multiple bands by SSCP and necessitated addition of this technique to degenerate RT-PCR for further analysis.
Conclusion: It could be suggested that degenerate RT-PCR/SSCP is practical method and could be used as a screening test for transcriptome profiling of various disease states with further validation study.
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KEYWORD
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RT-PCR/SSCP, Transcriptome profiling, TNF-a, 3T2-L1 adipocyte
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