An integrated technique for identification of differential genes expressed in patients with cancer

Abstract

To develop a method for identification of differential gene expression between different cell population, several conveninent techniques of molecular biology, including subtractive hybridization, suppression PCR, T/A cloning and sequencing, were used to identify genes expressed differentially in CD45⁻ and CD45⁻ cells isolated from U266 cell line of multiple myeloma. Our results showed that the levels of abundant genes genes scale down 20 times through subtractive hybridization. Plasmid DNA from CD45 cell clones was hybridized with forward or backward cDNA probes synthesized from CD45 and CD45 cells, respectively. A few of differentially expressed genes reconfirmed by RT-PCR were identified from 500 expressed clones of CD45′ cells. It is concluded that a strategy for gene expression identification developed from conventional molecular biological methods can be used in different laboratories.

Keywords

gene expression / subtractive hybridization / differential display

References

[1] ZhangL, ZhouW, VelculescuV E, et al. . Gene expression profiles in normal and cancer cells. Science, 1997, 276: 1268-1268

[2] MahmoudM S, IshikawaH, FujiiR, et al. . Induction of CD45 expression and proliferation in U266 myeloma cell line by interleukin 6. Blood, 1998, 92: 3887-3887

[3] DiatchenkoL, LauY C, CampbellA P, et al. . Supression subtractive hybridization: A method for generating differentially regulation or tissue-specific cDNA probes and libraries. Proc Natl Acad Sci USA, 1996, 93: 6025-6025

[4] TravisG H, SutcliffeJ G. Phenol emulsion-enhanced DNA-driven subtractive cDNA cloning: isolation of lowabundance monkey cortex-specific mRNAs. Proc. Natl Acad Sci USA, 1988, 85: 1696-1696

[5] LiangP, PardeeA B. Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction. Science, 1992, 257: 967-967

[6] VelculescuV E, ZhangL, VogelsteinB, et al. . Serial analysis of gene expression. Science, 1995, 270: 484-484

[7] DanielJ L, LuisL M, FabienneF O, et al. . Selective amplification via biotin- and restriction-mediated enrichment (SABRE), a novel selective amplification procedure for detection of differentially expressed mRNA. Proc Natl Acad Sci USA, 1997, 94: 6831-6831

[8] San MingW, JanetD R. A strategy for genome-wide analysis: Integrated procedure for gene identification. Proc Natl Acad Sci USA, 1998, 95: 11909-11909