Identification and isolation of differentially expressed genes from very small tissue samples.

Published

Journal Article

Identification of differentially expressed genes from tissue samples weighing only a few milligrams has remained a major challenge. Here, we describe a novel and simple strategy that uses standard molecular biology equipment and commercially available kits. The approach combines isolation of total RNA by silica-gel binding, reverse transcription using anchored modified, 5' end enhancers oligonucleotides, exponential amplification of the single-stranded cDNA and hybridization to high-density cDNA filter arrays. The method was tested by comparing genes expressed on freshly isolated human trabecular meshwork tissue with those expressed in corresponding primary cells at third passage. Validation was achieved by using two biological properties: (i) hybridization, to identify the differentially expressed genes, and (ii) PCR amplification, to confirm their distinct expression. The strategy presented allows the identification of differentially expressed genes and/or uncharacterized expressed sequence tags (ESTs) in very small tissue samples, including those from clinical specimens.

Full Text

Duke Authors

Cited Authors

  • Gonzalez, P; Zigler, JS; Epstein, DL; Borrás, T

Published Date

  • May 1999

Published In

Volume / Issue

  • 26 / 5

Start / End Page

  • 884 - 892

PubMed ID

  • 10337481

Pubmed Central ID

  • 10337481

Electronic International Standard Serial Number (EISSN)

  • 1940-9818

International Standard Serial Number (ISSN)

  • 0736-6205

Digital Object Identifier (DOI)

  • 10.2144/99265st01

Language

  • eng