Identification of novel bovine RPE and retinal genes by subtractive hybridization

Shiwani Sharma, Jinghua T. Chang, Neil G. Della, Peter A Campochiaro, Donald J Zack

Research output: Contribution to journalArticle

Abstract

Purpose: Understanding of the specialized function of the retinal pigment epithelium (RPE) can be aided by the identification and characterization of genes that are preferentially expressed in the RPE. With this aim, we undertook a systematic effort to identify and begin characterization of such genes. Methods: A subtracted bovine RPE cDNA library was generated through subtractive hybridization using a single-stranded circular bovine RPE cDNA library as target and biotinylated mRNA from bovine heart and liver as alternate drivers. Approximately one thousand of the resulting subtracted cDNA clones were partially sequenced and analyzed, and a non-redundant set of one hundred of these cDNAs were examined for tissue expression pattern using a mini-Northern blot procedure and for identity by sequence analysis. Results: The subtraction method successfully allowed the enrichment of cDNAs that are preferentially expressed in the RPE. Out of the analyzed clones, expression of forty-five clones was verifiable by Northern blotting. Of these, a significant proportion of cDNAs were preferentially expressed in the RPE. We observed that the expression of some subtracted cDNAs was restricted to the retina and no expression was detected in the RPE. These retinal clones were obtained in addition to RPE clones presumably because the initial RPE RNA population was contaminated with a small proportion of retinal RNA. Two thirds of the identified RPE and retinal cDNAs are likely to represent novel genes because they do not have homology to known genes in the databases. Conclusions: Genes that are specifically or predominantly expressed in the RPE/retina are likely to be important for retinal function. We have identified novel cDNAs from bovine RPE and retina by subtractive hybridization. These cDNAs can be used as starting material for the identification of corresponding human genes expressed in the RPE and retina. The human genes thus identified are likely to contain good candidate genes for retinal disease.

Original languageEnglish (US)
Pages (from-to)251-258
Number of pages8
JournalMolecular Vision
Volume8
StatePublished - 2002

Fingerprint

Retinal Pigment Epithelium
Complementary DNA
Genes
Clone Cells
Retina
Gene Library
Northern Blotting
Subtractive Hybridization Techniques
RNA
Retinal Diseases
Forensic Anthropology
Sequence Analysis

ASJC Scopus subject areas

  • Ophthalmology

Cite this

Identification of novel bovine RPE and retinal genes by subtractive hybridization. / Sharma, Shiwani; Chang, Jinghua T.; Della, Neil G.; Campochiaro, Peter A; Zack, Donald J.

In: Molecular Vision, Vol. 8, 2002, p. 251-258.

Research output: Contribution to journalArticle

@article{a5e7fd4dae5447e38cab35fccab71ce2,
title = "Identification of novel bovine RPE and retinal genes by subtractive hybridization",
abstract = "Purpose: Understanding of the specialized function of the retinal pigment epithelium (RPE) can be aided by the identification and characterization of genes that are preferentially expressed in the RPE. With this aim, we undertook a systematic effort to identify and begin characterization of such genes. Methods: A subtracted bovine RPE cDNA library was generated through subtractive hybridization using a single-stranded circular bovine RPE cDNA library as target and biotinylated mRNA from bovine heart and liver as alternate drivers. Approximately one thousand of the resulting subtracted cDNA clones were partially sequenced and analyzed, and a non-redundant set of one hundred of these cDNAs were examined for tissue expression pattern using a mini-Northern blot procedure and for identity by sequence analysis. Results: The subtraction method successfully allowed the enrichment of cDNAs that are preferentially expressed in the RPE. Out of the analyzed clones, expression of forty-five clones was verifiable by Northern blotting. Of these, a significant proportion of cDNAs were preferentially expressed in the RPE. We observed that the expression of some subtracted cDNAs was restricted to the retina and no expression was detected in the RPE. These retinal clones were obtained in addition to RPE clones presumably because the initial RPE RNA population was contaminated with a small proportion of retinal RNA. Two thirds of the identified RPE and retinal cDNAs are likely to represent novel genes because they do not have homology to known genes in the databases. Conclusions: Genes that are specifically or predominantly expressed in the RPE/retina are likely to be important for retinal function. We have identified novel cDNAs from bovine RPE and retina by subtractive hybridization. These cDNAs can be used as starting material for the identification of corresponding human genes expressed in the RPE and retina. The human genes thus identified are likely to contain good candidate genes for retinal disease.",
author = "Shiwani Sharma and Chang, {Jinghua T.} and Della, {Neil G.} and Campochiaro, {Peter A} and Zack, {Donald J}",
year = "2002",
language = "English (US)",
volume = "8",
pages = "251--258",
journal = "Molecular Vision",
issn = "1090-0535",

}

TY - JOUR

T1 - Identification of novel bovine RPE and retinal genes by subtractive hybridization

AU - Sharma, Shiwani

AU - Chang, Jinghua T.

AU - Della, Neil G.

AU - Campochiaro, Peter A

AU - Zack, Donald J

PY - 2002

Y1 - 2002

N2 - Purpose: Understanding of the specialized function of the retinal pigment epithelium (RPE) can be aided by the identification and characterization of genes that are preferentially expressed in the RPE. With this aim, we undertook a systematic effort to identify and begin characterization of such genes. Methods: A subtracted bovine RPE cDNA library was generated through subtractive hybridization using a single-stranded circular bovine RPE cDNA library as target and biotinylated mRNA from bovine heart and liver as alternate drivers. Approximately one thousand of the resulting subtracted cDNA clones were partially sequenced and analyzed, and a non-redundant set of one hundred of these cDNAs were examined for tissue expression pattern using a mini-Northern blot procedure and for identity by sequence analysis. Results: The subtraction method successfully allowed the enrichment of cDNAs that are preferentially expressed in the RPE. Out of the analyzed clones, expression of forty-five clones was verifiable by Northern blotting. Of these, a significant proportion of cDNAs were preferentially expressed in the RPE. We observed that the expression of some subtracted cDNAs was restricted to the retina and no expression was detected in the RPE. These retinal clones were obtained in addition to RPE clones presumably because the initial RPE RNA population was contaminated with a small proportion of retinal RNA. Two thirds of the identified RPE and retinal cDNAs are likely to represent novel genes because they do not have homology to known genes in the databases. Conclusions: Genes that are specifically or predominantly expressed in the RPE/retina are likely to be important for retinal function. We have identified novel cDNAs from bovine RPE and retina by subtractive hybridization. These cDNAs can be used as starting material for the identification of corresponding human genes expressed in the RPE and retina. The human genes thus identified are likely to contain good candidate genes for retinal disease.

AB - Purpose: Understanding of the specialized function of the retinal pigment epithelium (RPE) can be aided by the identification and characterization of genes that are preferentially expressed in the RPE. With this aim, we undertook a systematic effort to identify and begin characterization of such genes. Methods: A subtracted bovine RPE cDNA library was generated through subtractive hybridization using a single-stranded circular bovine RPE cDNA library as target and biotinylated mRNA from bovine heart and liver as alternate drivers. Approximately one thousand of the resulting subtracted cDNA clones were partially sequenced and analyzed, and a non-redundant set of one hundred of these cDNAs were examined for tissue expression pattern using a mini-Northern blot procedure and for identity by sequence analysis. Results: The subtraction method successfully allowed the enrichment of cDNAs that are preferentially expressed in the RPE. Out of the analyzed clones, expression of forty-five clones was verifiable by Northern blotting. Of these, a significant proportion of cDNAs were preferentially expressed in the RPE. We observed that the expression of some subtracted cDNAs was restricted to the retina and no expression was detected in the RPE. These retinal clones were obtained in addition to RPE clones presumably because the initial RPE RNA population was contaminated with a small proportion of retinal RNA. Two thirds of the identified RPE and retinal cDNAs are likely to represent novel genes because they do not have homology to known genes in the databases. Conclusions: Genes that are specifically or predominantly expressed in the RPE/retina are likely to be important for retinal function. We have identified novel cDNAs from bovine RPE and retina by subtractive hybridization. These cDNAs can be used as starting material for the identification of corresponding human genes expressed in the RPE and retina. The human genes thus identified are likely to contain good candidate genes for retinal disease.

UR - http://www.scopus.com/inward/record.url?scp=0347265312&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0347265312&partnerID=8YFLogxK

M3 - Article

C2 - 12131876

AN - SCOPUS:0347265312

VL - 8

SP - 251

EP - 258

JO - Molecular Vision

JF - Molecular Vision

SN - 1090-0535

ER -