A cDNA RDA protocol using solid-phase technology suited for analysis in small tissue samples.
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.
Odeberg
J
J
Department of Biotechnology, KTH, Royal Institute of Technology, Stockholm, Sweden.
Wood
T
T
Blücher
A
A
Rafter
J
J
Norstedt
G
G
Lundeberg
J
J
D000180:Acrylic Resins / Q000737:chemistry; D000818:Animals; D001706:Biopsy; D003001:Cloning, Molecular; D003110:Colonic Neoplasms / Q000235:genetics; D017931:DNA Primers / Q000737:chemistry; D018076:DNA, Complementary / Q000737:chemistry* / Q000378:metabolism; D004273:DNA, Neoplasm / Q000737:chemistry*; D004587:Electrophoresis, Agar Gel; D013006:Growth Hormone / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D008099:Liver / Q000187:drug effects; D008297:Male; D009693:Nucleic Acid Hybridization / Q000379:methods; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000032:analysis / Q000378:metabolism; D051381:Rats; D013382:Subtraction Technique; D014020:Tissue Extracts
D000180:Acrylic Resins; D017931:DNA Primers; D018076:DNA, Complementary; D004273:DNA, Neoplasm; D012333:RNA, Messenger; D014020:Tissue Extracts; C077041:Bio acrylic resin; D013006:Growth Hormone