Jiang H, Kang D C, Alexandre D, Fisher P B
Departments of Pathology, Urology, and Neurosurgery, Herbert Irving Comprehensive Cancer Center, Columbia University, College of Physicians and Surgeons, New York, NY 10032, USA.
Proc Natl Acad Sci U S A. 2000 Nov 7;97(23):12684-9. doi: 10.1073/pnas.220431297.
Human melanoma cells growth-arrest irreversibly and terminally differentiate on treatment with a combination of fibroblast interferon and the protein kinase C activator mezerein. This experimental protocol also results in a loss of tumorigenic potential and profound changes in gene expression. Various cloning and cDNA microarray strategies are being used to determine the complete spectrum of gene expression changes underlying these alterations in human melanoma cells. An efficient approach, Rapid Subtraction Hybridization (RaSH), has been developed that is permitting the identification of genes of potential relevance to cancer growth control and terminal cell differentiation. RaSH cDNA libraries are prepared from double-stranded cDNAs that are enzymatically digested into small fragments, ligated to adapters, and PCR amplified followed by incubation of tester and driver PCR fragments. This subtraction hybridization scheme is technically simple and results in the identification of a high proportion of differentially expressed sequences, including known genes and those not described in current DNA databases. The RaSH approach represents an efficient methodology for identifying and cloning genes displaying differential expression that associate with and potentially regulate complex biological processes.
人黑色素瘤细胞在用成纤维细胞干扰素和蛋白激酶C激活剂芫花酯素联合处理后会不可逆地生长停滞并终末分化。该实验方案还会导致致瘤潜能丧失和基因表达的深刻变化。各种克隆和cDNA微阵列策略正被用于确定人黑色素瘤细胞这些改变背后基因表达变化的完整谱。一种有效的方法，即快速扣除杂交（RaSH）已被开发出来，它能够鉴定与癌症生长控制和终末细胞分化潜在相关的基因。RaSH cDNA文库是由双链cDNA制备而成，双链cDNA被酶切为小片段，连接上接头，进行PCR扩增，然后将测试者和驱动者的PCR片段进行孵育。这种扣除杂交方案技术上很简单，能鉴定出高比例的差异表达序列，包括已知基因和当前DNA数据库中未描述的基因。RaSH方法是一种用于鉴定和克隆显示差异表达且与复杂生物学过程相关并可能对其进行调控的基因的有效方法。
