A cDNA RDA protocol using solid-phase technology suited for analysis in small tissue samples.
2000 (English)In: Biomolecular Engineering, ISSN 1389-0344, E-ISSN 1878-559X, Vol. 17, no 1, 1-9 p.Article in journal (Refereed) Published
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.
IdentifiersURN: urn:nbn:se:kth:diva-13363PubMedID: 11042471OAI: oai:DiVA.org:kth-13363DiVA: diva2:324420
QC 201006152010-06-152010-06-152010-06-17Bibliographically approved