Discovery of differentially expressed genes in human breast cancer using subtracted cDNA libraries and cDNA microarrays

Abstract

Identifying novel and known genes that are di erentially expressed in breast cancer has important implications in understanding the biology of breast tumorigenesis and developing new diagnostic and therapeutic agents. In this study we have combined two powerful technologies, PCRbased cDNA subtraction and cDNA microarray, as a high throughput methodology designed to identify cDNA clones that are breast tumor-and tissue-speci®c and are overexpressed in breast tumors. Approximately 2000 cDNA clones generated from the subtracted breast tumor library were arrayed on the microarray chips. The arrayed target cDNAs were then hybridized with 30 pairs of¯uorescent-labeled cDNA probes generated from breast tumors and normal tissues to determine the tissue distribution and tumor speci®city. cDNA clones showing overexpression in breast tumors by microarray were further analysed by DNA sequencing, GenBank and EST database searches, and quantitative real time PCR. We identi®ed several known genes, including mammaglobin, cytokeratin 19, ®bronectin, and hair-speci®c type II keratin, which have previously been shown to be overexpressed in breast tumors and may play an important role in the malignance of breast. We also discovered B726P which appears to be an isoform of NY-BR-1, a breast tissue-speci®c gene. Two additional clones discovered, B709P and GABA A receptor p subunit, were not previously described for their overexpression pro®le in breast tumors. Thus, combining PCRbased cDNA subtraction and cDNA microarray allowed for an e cient way to identify and validate genes with elevated mRNA expression levels in breast cancer that may potentially be involved in breast cancer progression. These di erentially expressed genes may be of potential utility as therapeutic and diagnostic targets for breast cancer.

Identification of differentially expressed genes in clear cell renal cell carcinoma by analysis of full-length enriched cDNA library

“…This method provides more precise measurement of gene expression than the technology of cDNA microarray, which is usually performed to identify differentially expressed genes [15,16], and more convenient acquirement of the full-length cDNAs for further studying than the analysis of cDNA subtraction [17,18]. In this study, we identified a group of up-or down-regulated genes by analysis of full-length cDNA libraries, and found significant variations between the gene expression profiles of ccRCC and normal kidney tissues.…”

HGF induces fibronectin matrix synthesis in melanoma cells through MAP kinase-dependent signaling pathway and induction of Egr-1

“…Reduction or loss of fibronectin expression has been observed in many transformed cells in culture (Ruoslahti and Giancotti, 1989). However, overexpression of fibronectin is associated with various human tumor cells including colorectal cancer, breast carcinoma, head and neck squamous carcinoma and metastatic melanoma (Zhang et al, 1997;Bittner et al, 2000;Al Moustafa et al, 2002;Jiang et al, 2002).…”

A Novel mRNA Level Subtraction Method for Quick Identification of Target-Orientated Uniquely Expressed Genes Between Peanut Immature Pod and Leaf

“…This library clone capacity was much lesser than those of serial subtraction [5,13] or standard PCR-based cDNA subtraction protocols, which often generate libraries of capacities ranging from a few thousands to more than 10,000 clones [4,8,11,12]. Totally, 46 clones from plating assays were randomly selected for 5'-end single-pass DNA sequencing.…”

“…(c) The protocol does not require PCR cloning for subtractive library construction. This is beneficial because it prevents developing subtractive libraries with larger capacities of clones as previously reported [4,5,8,11-13]. (d) There is no need for radioisotopes and/or some related specialized enzymes such as [α- 32 P]dCTP [11,14] and terminal transferase [11].…”

“…This limits rapid and more efficient detections of uniquely expressed target gene(s) within a defined biological material or process [5,7]. Such challenge coupled generally with the larger clone capacity of the first subtraction library had inspired current subtraction protocol modifications [4,12], among which some have involved a few to extended serial subtractions [5,8,13], whereas the others had combined both subtraction and microarray hybridization [4,7,9] for determining target specific genes. However, this usually is tedious and entails technical complexity and increased reagent and labor costs while also extending the experimental period.…”