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A cDNA RDA protocol using solid-phase technology suited for analysis in small tissue samples
KTH, Superseded Departments, Biotechnology.
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2000 (English)In: Biomolecular Engineering, ISSN 1389-0344, E-ISSN 1878-559X, Vol. 17, no 1, 1-9 p.Article in journal (Refereed) Published
Abstract [en]

cDNA representational difference analysis (cDNA RDA) is a PCR-based subtractive enrichment procedure for the cloning of differentially expressed genes. In this study, we have further developed the procedure to take advantage of solid-phase technology, and to facilitate the use of RDA when starting material is limited. Several parameters of the PCR-based generation of cDNA representations were investigated, and a solid-phase based purification step was introduced to simplify removal of digested adapter-ends and uncleaved fragments. The use of magnetic particles increased the speed of the method, and also eliminated the risk of carry-over contamination between iterative steps of subtraction and PCR amplification. The modified protocol was evaluated in monitoring differences in gene expression in (i) a rat system consisting of livers with and without growth hormone treatment, and in (ii) a human system consisting of normal colon and colon cancer.

Place, publisher, year, edition, pages
2000. Vol. 17, no 1, 1-9 p.
Keyword [en]
cDNA, representational difference analysis (RDA), solid-phase, PCR, subtractive cloning, representational difference analysis, arbitrarily primed pcr, expressed genes, messenger-rnas, display, hybridization, dna, identification, cloning
URN: urn:nbn:se:kth:diva-20108ISI: 000089919900001OAI: diva2:338801
QC 20100525Available from: 2010-08-10 Created: 2010-08-10Bibliographically approved

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