Identification and isolation of differentially expressed genes from very small tissue samples.
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.
Gonzalez
P
P
Duke University Medical Center, Durham, NC 27710, USA.
Zigler
J S
JS
Epstein
D L
DL
Borrás
T
T
D001709:Biotechnology; D002478:Cells, Cultured; D018076:DNA, Complementary; D020224:Expressed Sequence Tags; D005353:Fibronectins / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D014018:Tissue Distribution; D014129:Trabecular Meshwork / Q000378:metabolism
D018076:DNA, Complementary; D005353:Fibronectins; D012333:RNA, Messenger; D007041:Hypoxanthine Phosphoribosyltransferase