Isolation of human ear specific cDNAs and construction of cDNA libraries from surgically removed small amounts of inner ear tissues

Abstract

We have used representational difference analysis (RDA) for subtractive hybridization of oligo dT primed directionally cloned cDNA libraries from human inner ear tissue and a B-lymphoblast cell line. Two rounds of subtraction-amplification, followed by differential hybridization of selected clones led to the isolation of genes which were specific to the ear. Sequence analysis of randomly chosen clones revealed the presence of a histidine rich Ca2+ binding protein, human dynamin, collagen type 1A1, collagen type 2A1, SPARC, human growth hormone, and several specific genes which had no sequence homology in the data base. Furthermore, to apply these techniques for isolating genes specific to distinct inner ear structures and/or cell types of inner ear for which the starting tissue material is limiting, we have used a modified PCR based protocol to construct representative cDNA libraries. We have characterized a cDNA library constructed from small amounts of inner ear tissues recovered by ablative surgical procedure involving labyrinthectomy. The potential application of these protocols for isolating genes involved in hearing and deafness is discussed.

Gene expression signatures and biomarkers of noninvasive and invasive breast cancer cells: comprehensive profiles by representational difference analysis, microarrays and proteomics

“…The RDA protocol is powerful enough to yield important genes that show significant alterations in their expression between cell lines, and can lead to isolation of full-length cDNAs by using appropriate modifications (Baskaran et al, 1996;Jacob et al, 1997). The detection of RUNX2, variant of RUNX2, EphB6, prion protein, lysyl oxidase and a copper transporter ATP7A transcripts by RDA warrant specific mention.…”

“…In the second experiment, MCF-7 cDNA was used as a tester and MDA-MB-231 cDNA as a driver. The protocol has been describer previously (Jacob et al, 1997). Briefly, first-strand synthesis was carried out using a commercial cDNA synthesis kit as per the manufacturer's protocol.…”

Expression of mRNA encoding extracellular matrix glycoproteins SPARC and SC1 is temporally and spatially regulated in the developing cochlea of the rat inner ear

“…Recently there has been much interest in the characterization of cochlea-speci¢c genes in order to facilitate the identi¢cation of candidate genes for human hearing disorders and to increase the understanding of auditory function at the molecular level. Sequence analysis of randomly chosen clones from human fetal inner ear or cochlear cDNA libraries has revealed an abundance of signal transduction proteins, tra¤cking proteins and components of the ECM (Robertson et al, 1994;Jacob et al, 1997 ;Skvorak et al, 1999). A number of ECM genes are expressed in the inner ear, serving structural or regulatory functions.…”

“…Interestingly, SPARC is one of the most highly expressed genes in the human fetal cochlea Jacob et al, 1997;Skvorak et al, 1999). SPARC is expressed in tissues undergoing reorganization and is thought to modulate cell^matrix interactions by inhibiting cell spreading and cell cycle progression and by regulating the production of other ECM molecules (Motamed, 1999).…”

“…Mutations a¡ecting matrix molecules cause inherited forms of inner ear disease (Petit, 1996;Corwin, 1998;Cremers, 1999 ;McGuirt et al, 1999 ;Steel and Bussoli, 1999). Analysis of human fetal cochlear cDNA libraries has shown that in addition to collagen, SPARC is one of the most highly expressed genes in the developing cochlea (Robertson et al, 1994;Jacob et al, 1997;Skvorak et al, 1999).…”