Fluorescent differential display analysis of gene expression in differentiating neuroblastoma cells.
Identification of differentially-expressed genes provides an important step toward the elucidation of molecular mechanisms underlying a variety of biological processes. A novel PCR-based approach to detect and clone such transcripts is the so-called differential display (DD). We established an improved DD protocol that can be performed on an automated fluorescent DNA sequencer to ensure high throughput as well as operational safety. Using this fluorescent DD (FDD) technique, we analyzed the gene expression profile in the retinoic acid-induced differentiation of a human neuroblastoma cell line SH-SY5Y. Screening with 102 primer combinations at eight different time points revealed 66 cDNA bands with variously different behaviors out of approximately 6000 bands displayed. Subsequent analyses with 15 cloned species confirmed the differential expression of corresponding transcripts in all the cases, thereby demonstrating the high reliability of FDD analysis. These clones were composed of seven novel and eight known genes, the latter of which included those that had never been described in the context of neuronal differentiation. These results indicate that FDD is an effective approach to obtain not only novel genes but also clues to possible novel functions of known genes involved in various biological phenomena.
Kito
K
K
Human Genome Center, Institute of Medical Science, University of Tokyo, Minato-ku, Japan.
Ito
T
T
Sakaki
Y
Y
D000595:Amino Acid Sequence; D001483:Base Sequence; D015152:Blotting, Northern; D002454:Cell Differentiation / Q000235:genetics*; D002999:Clone Cells; D003001:Cloning, Molecular; D017931:DNA Primers; D018076:DNA, Complementary / Q000737:chemistry; D019793:Fluorescein; D005452:Fluoresceins / Q000378:metabolism; D005453:Fluorescence; D015972:Gene Expression Regulation, Neoplastic / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D009447:Neuroblastoma; D016133:Polymerase Chain Reaction; D014158:Transcription, Genetic / Q000235:genetics; D014407:Tumor Cells, Cultured
D017931:DNA Primers; D018076:DNA, Complementary; D005452:Fluoresceins; D019793:Fluorescein