J Korean Diabetes Assoc.  2007 Sep;31(5):410-420.

Transcription Factor Profile by Degenerate RT-PCR/SSCP: Application in 3T3-L1 Adipocyte Treated with TNF-alpha

• Kim YL
• Lee SH
• Choi YK
• Chang SY
• Kim YS
• Kim SK
• Park SW
• Park WK
• Cho YW
• Lee SJ

Abstract

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.

Keyword

RT-PCR (reverse transcriptase polymerase-chain reaction)/SSCP (single strand conformational polymorphism); Transcriptome profiling; TNF-alpha; 3T3-L1 adipocyte

MeSH Terms

Adipocytes*
Electrophoresis
Gene Expression
Gene Expression Profiling
Mass Screening
Oligonucleotide Array Sequence Analysis
Polymerase Chain Reaction
Polymorphism, Single-Stranded Conformational
RNA, Messenger
Sepharose
Transcription Factors*
Transcriptome
Tumor Necrosis Factor-alpha*
RNA, Messenger
Sepharose
Transcription Factors
Tumor Necrosis Factor-alpha

Figure

• 

  Fig. 1 Effect of TNF-α on protein level of insulin receptor substrate 1 (IRS-1) in 3T3-L1 adipocytes by gel electrophoresis and immunoblotting (Ctrl: 3T3-L1 preadipocyte, lane 1: fully differentiated 3T3-L1 adipocyte, lane 2: adipocyte treated with 100 nM insulin for 15 min, lane 3: adipocyte treated with 3 ng/mL TNF-α for 48 h, lane 4: adipocyte treated with TNF-α and insulin).

• 

  Fig. 2 Agarose gel electrophoresis of transcription factor degenerate RT-PCRs on total RNAs of fully differentiated 3T3-L1 adipocytes before and after TNF-α treatment. Numbers above the columns mean categorized groups of transcription factors. Each left and right column below each number shows the result of RT-PCR from 3T3-L1 adipocytes before and after TNF-α treatment, respectively. Columns without number on the top are markers for the sizes (base pair) of PCR product.

• 

  Fig. 3 SSCP (single strand conformational polymorphism) analysis of transcription factor RT-PCRs. The products, taken from 28 and 75 RT-PCRs in Fig. 2 were denatured at 96℃ for 5min, cooled in ice for 5 min, and then 3 times loaded on 12% nondenaturing polyacrylamide gel.

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