Combining SSH and cDNA microarrays for rapid identification of differentially expressed genes.
Comparing patterns of gene expression in cell lines and tissues has important applications in a variety of biological systems. In this study we have examined whether the emerging technology of cDNA microarrays will allow a high throughput analysis of expression of cDNA clones generated by suppression subtractive hybridization (SSH). A set of cDNA clones including 332 SSH inserts amplified by PCR was arrayed using robotic printing. The cDNA arrays were hybridized with fluorescent labeled probes prepared from RNA from ER-positive (MCF7 and T47D) and ER-negative (MDA-MB-231 and HBL-100) breast cancer cell lines. Ten clones were identified that were over-expressed by at least a factor of five in the ER-positive cell lines. Northern blot analysis confirmed over-expression of these 10 cDNAs. Sequence analysis identified four of these clones as cytokeratin 19, GATA-3, CD24 and glutathione-S-transferase mu-3. Of the remaining six cDNA clones, four clones matched EST sequences from two different genes and two clones were novel sequences. Flow cytometry and immunofluorescence confirmed that CD24 protein was over-expressed in the ER-positive cell lines. We conclude that SSH and microarray technology can be successfully applied to identify differentially expressed genes. This approach allowed the identification of differentially expressed genes without the need to obtain previously cloned cDNAs.
Department of Surgery and Department of Biochemistry, Stanford University, Stanford, CA 94305-5414, USA.
G P
GP
Yang
D T
DT
Ross
W W
WW
Kuang
P O
PO
Brown
R J
RJ
Weigel
D015703:Antigens, CD / Q000235:genetics / Q000302:isolation & purification; D015152:Blotting, Northern; D001943:Breast Neoplasms / Q000235:genetics*; D051927:CD24 Antigen; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004268:DNA-Binding Proteins / Q000235:genetics; D020224:Expressed Sequence Tags; D005260:Female; D050990:GATA3 Transcription Factor; D015870:Gene Expression; D015723:Gene Library; D005982:Glutathione Transferase / Q000235:genetics; D006801:Humans; D007633:Keratins / Q000235:genetics; D008562:Membrane Glycoproteins; D009693:Nucleic Acid Hybridization / Q000379:methods*; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D012333:RNA, Messenger / Q000302:isolation & purification*; D011960:Receptors, Estrogen / Q000302:isolation & purification; D017422:Sequence Analysis, DNA; D015534:Trans-Activators / Q000235:genetics; D014407:Tumor Cells, Cultured
D015703:Antigens, CD; D051927:CD24 Antigen; C497889:CD24 protein, human; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D050990:GATA3 Transcription Factor; C494749:GATA3 protein, human; D008562:Membrane Glycoproteins; D012333:RNA, Messenger; D011960:Receptors, Estrogen; D015534:Trans-Activators; D007633:Keratins; D005982:Glutathione Transferase