A high throughput screening for rarely transcribed differentially expressed genes.
A novel method combining elements of suppression subtractive hybridization with high throughput differential screening permits the efficient and rapid cloning of rarely transcribed differentially expressed genes. The experimental strategy virtually excludes the possibility of isolating false positive clones. The potential of the method is demonstrated by the isolation of 625 differentially expressed cDNAs from the metastatic adenocarcinoma cell line Bsp73-ASML when subtracted from its non-metastatic counterpart Bsp73-1AS. Northern analysis of 72 randomly selected clones demonstrated that 68 were differentially expressed with respect to Bsp73-ASML, indicating a true positive rate of 94%. Additionally, a large proportion of these clones represented rare transcripts as determined by the exposure time required to detect a signal. Sequence data indicated that of the 625 clones obtained, 92 clones scored perfect or near perfect matches with already known genes. Two hundred and eighty one clones scored between 60 and 95% homology to known human and mouse genes, whereas 252 clones scored no match with any sequences in the public databases. The method we describe is ideally suited whenever subtle changes in gene expression profiles need to be determined.
von Stein
O D
OD
Institut für Genetik (IGEN) and 1 Institute für Toxikologie (ITOX), Forschungszentrum Karlsruhe, PO Box 3640, 76021 Karlsruhe, Germany.
Thies
W G
WG
Hofmann
M
M
D000230:Adenocarcinoma / Q000235:genetics*; D000818:Animals; D015152:Blotting, Northern; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry / Q000302:isolation & purification; D004273:DNA, Neoplasm / Q000737:chemistry / Q000302:isolation & purification; D015723:Gene Library; D006801:Humans; D051379:Mice; D009362:Neoplasm Metastasis; D009693:Nucleic Acid Hybridization / Q000379:methods*; D010190:Pancreatic Neoplasms / Q000235:genetics*; D016133:Polymerase Chain Reaction; D051381:Rats; D017385:Sequence Homology; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured; D014568:Urokinase-Type Plasminogen Activator / Q000235:genetics
D018076:DNA, Complementary; D004273:DNA, Neoplasm; D014568:Urokinase-Type Plasminogen Activator