Efficacy of SSHPCR in isolating differentially expressed genes

被引:58
作者
Ji, W [1 ]
Wright, MB
Cai, L
Flament, A
Lindpaintner, K
机构
[1] Harvard Univ, Brigham & Womens Hosp, Dept Cardiovasc Med, Sch Med, Boston, MA 02115 USA
[2] F Hoffmann La Roche & Co Ltd, Roche Genet, CH-4002 Basel, Switzerland
[3] F Hoffmann La Roche & Co Ltd, Dept Vasc & Metab Dis, CH-4002 Basel, Switzerland
关键词
D O I
10.1186/1471-2164-3-12
中图分类号
Q81 [生物工程学(生物技术)]; Q93 [微生物学];
学科分类号
071005 ; 0836 ; 090102 ; 100705 ;
摘要
Background: Suppression Subtractive Hybridization PCR (SSH PCR) is a sophisticated cDNA subtraction method to enrich and isolate differentially expressed genes. Despite its popularity, the method has not been thoroughly studied for its practical efficacy and potential limitations. Results: To determine the factors that influence the efficacy of SSH PCR, a theoretical model, under the assumption that cDNA hybridization follows the ideal second kinetic order, is proposed. The theoretical model suggests that the critical factor influencing the efficacy of SSH PCR is the concentration ratio (R) of a target gene between two cDNA preparations. It preferentially enriches "all or nothing" differentially expressed genes, of which R is infinite, and strongly favors the genes with large R. The theoretical predictions were validated by our experiments. In addition, the experiments revealed some practical limitations that are not obvious from the theoretical model. For effective enrichment of differentially expressed genes, it requires fractional concentration of a target gene to be more than 0.01 % and concentration ratio to be more than 5 folds between two cDNA preparations. Conclusion: Our research demonstrated theoretical and practical limitations of SSH PCR, which could be useful for its experimental design and interpretation.
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页数:7
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