Subtractive hybridization – genetic takeaways and the search for meaning

Abstract

Gene expression profiling relies on mRNA extraction from defined cell systems, which in the case of pathological processes necessarily results in the use of small quantities of tissues, sometimes as little as a few cells. This obviates the use of many systems of gene expression profiling and is best carried out using cDNA amplified by poly(A) reverse transcription polymerase chain reaction, which is capable of generating material representative of all the expressed genes in samples as small as one cell. Analysis of this material using subtractive hybridization compares the genes expressed at different stages of a biological/pathological process allowing identification of the all the genes upregulated during the process. The identification of the genes present is not dependent on their prior description or on the choice of genes used in a screen and as such the method is ideal for identifying novel genes or unsuspected genes. We have used the method to identify genes involved in normal osteoblastic differentiation and in Paget's disease of bone and it has been widely used to study normal differentiation and pathological processes in a number of systems. The method, its applications and its relationship with the other methods of gene expression profiling are reviewed.

Syntenin is overexpressed and promotes cell migration in metastatic human breast and gastric cancer cell lines

“…Various methods for comparing patterns of gene expression have been described in the literature (Byers et al, 2000). Of these, subtractive suppression hybridization (SSH) represents a simple and e cient technique for generating cDNAs that are highly enriched for di erentially expressed genes of both high and low abundance (Diatchenko et al, 1996).…”

Gene Expression Profiling in silico: Relative Expression of Candidate Angiogenesis Associated Genes in Renal Cell Carcinomas

“…Arrays enable the development of new hypotheses and potential paradigm shifts in our understanding of disease. The use of expression profiling by various means has been extensively reviewed in a number of recent publications [1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13]. In a recent review, we [14]discussed the use of these methods to discover genes associated with angiogenesis and compiled a list of growth factors, receptors, soluble molecules, and other genes that have been identified.…”

“…The von Hippel-Lindau (VHL) tumor suppressor gene is mutated in sporadic renal clear cell carcinomas [23]. The protein encoded by the VHL gene is part of an E3 ubiquitin-ligase complex and enables proteosomal degradation of the transcription factor, hypoxia- inducible factor 1 [1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24]. Mutations in VHL can thus result in elevated hypoxia-inducible factor 1 levels and upregulation of hypoxia-induced angiogenic genes such as VEGF.…”

Molecular Characterization of NF-HEV, a Nuclear Factor Preferentially Expressed in Human High Endothelial Venules

“…23,24 Nevertheless, such analysis is still hampered by the low number of cells available after purification, thereby ruling out traditional subtraction cloning techniques, which typically require several micrograms of mRNA. 25 To circumvent this problem, we previously adapted the PCR-based method of suppression subtractive hybridization (SSH) 26 to identify genes preferentially expressed in human tonsillar HEVECs compared with human umbilical vein endothelial cells (HUVECs). 27 With this method we generated a subtracted HEVEC cDNA library from 1 g of total RNA, and were able to clone several HEV-expressed cDNAs, including the promiscuous chemokine receptor DARC, mitochondrial genes, and secreted extracellular matrix (ECM) proteins, such as mac25/IGFBP7/angiomodulin.…”