Shotgun sequencing and microarray analysis of RDA transcripts.
Monitoring of differential gene expression is an important step towards understanding of gene function. We describe a comparison of the representational difference analysis (RDA) subtraction process with corresponding microarray analysis. The subtraction steps are followed in a quantitative manner using a shotgun cloning and sequencing procedure that includes over 1900 gene sequences. In parallel, the enriched transcripts are spotted onto microarrays facilitating large scale hybridization analysis of the representations and the difference products. We show by the shotgun procedure that there is a high diversity of gene fragments represented in the iterative RDA products (92-67% singletons) with a low number of shared sequences (<9%) between subsequent subtraction cycles. A non redundant set of 1141 RDA clones were immobilized on glass slides and the majority of these clones (97%) gave repeated good fluorescent signals in a subsequent hybridization of the labelled and amplified original cDNA. We observed only a low number of false positives (<2%) and a more than twofold differential expression for 32% (363) of the immobilized RDA clones. In conclusion, we show that by random sequencing of the difference products we obtained an accurate transcript profile of the individual steps and that large-scale confirmation of the obtained transcripts can be achieved by microarray analysis.
Andersson
Tove
T
Department of Biotechnology, Division of Molecular Biotechnology, KTH, Royal Institute of Technology, AlbaNova University Center, Stockholm Centre for Physics, Astronomy, and Biotechnology, SE-106 91, Stockholm, Sweden.
Boräng
Stina
S
Unneberg
Per
P
Wirta
Valtteri
V
Thelin
Anders
A
Lundeberg
Joakim
J
Odeberg
Jacob
J
D002460:Cell Line; D003001:Cloning, Molecular / Q000379:methods*; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D015536:Down-Regulation / Q000187:drug effects / Q000235:genetics; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D008077:Lipoproteins, LDL / Q000494:pharmacology; D020411:Oligonucleotide Array Sequence Analysis; D012333:RNA, Messenger / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D017422:Sequence Analysis, DNA; D014158:Transcription, Genetic / Q000187:drug effects; D015854:Up-Regulation / Q000187:drug effects / Q000235:genetics
D018076:DNA, Complementary; D008077:Lipoproteins, LDL; D012333:RNA, Messenger; C099252:oxidized low density lipoprotein