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

Identification of differentially expressed genes from tissue samples weighi ng only a few milligrams has remained a major challenge. Here, we describe a novel and simple strategy that rises 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-s tranded cDNA and hybridization to high-density cDNA filter arrays. The meth od was tested by comparing genes expressed on freshly isolated human trabec ular meshwork tissue with those expressed in corresponding primary cells at third passage. Validation,cas 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 pre sented allows the identification of differentially expressed genes and/or u ncharacterized expressed sequence tags (ESTs) in very small tissue samples, including those from clinical specimens.