Differential display, subtractive hybridization, and application of methodology to search for point mutations to identify genetic defects responsible for progression in MCF10AT model of human breast disease

摘要

We describe initial studies utilizing three methods to detect differences in gene expression: (i) differential display with polyT-anchored primers; (ii) differential display with RNA arbitrarily primed polymerase chain reaction (RAP-PCR), and (iii) cDNA subtractive hybridization. Subtractive hybridization, which detects qualitative differences in gene expression, revealed no differences between a human cell line (MCF-10A), orginated by spontaneous immortalization of breast epithelial cells, and MCF10CA1 cells, a recently derived fully malignant, metastatic variant. The standard method of differential display with polyT anchored primers detected in excess of 100 differentially displayed bands but differential expression could seldom be verified by Northern blotting. However, RAP-PCR products frequently represent the coding region and fewer bands are detected. One gene of interest is a zinc finger protein which may be expressed more in the preneoplastic lesion-forming cells than in nonlesion-forming cells. Because most bands are not consistently differentially displayed among the variants of the MCF10 model, we suspect that point mutations rather than differential quantitative gene expression is responsible for some stage of progression. We propose that differential display of RAP-PCR products on nondenaturing gels might also detect point mutation differences.