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

Pedro Gonzalez, J. Samuel Zigler, David L. Epstein, Teresa Borras

Research output: Contribution to journalArticlepeer-review

Abstract

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.

Original languageEnglish (US)
Pages (from-to)884-892
Number of pages9
JournalBioTechniques
Volume26
Issue number5
DOIs
StatePublished - 1999

ASJC Scopus subject areas

  • Biotechnology
  • Biochemistry, Genetics and Molecular Biology(all)

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