Reproducibility of targeted gene expression measurements in human islets of Langerhans.
The expression of 47 genes involved in the biosynthesis and secretion of insulin, apoptosis, and cellular stress was evaluated in isolated human islets using cDNA probes arrayed on nitrocellulose membranes. Isolated human islets were cultured for four days, or one month, with glucose present at a concentration of either 5.5 or 16.7 mmol/L. Extracted islet total RNA was used to generate [32P]dATP-labelled complex cDNA targets and hybridised with immobilised cDNA arrays. The positive expression of 45 mRNA transcripts in isolated human islets was documented. The coefficient of variance for relative levels of expression of transcripts was <25% for 9, 25-50% for 22, and 50-100% for 10, indicating good reproducibility between islet preparations from five different human pancreas donors. This study demonstrates the utility of nitrocellulose-based cDNA arrays for a focused reproducible analysis of gene expression changes in human islets of Langerhans.
Barry
Richard
R
Biochemistry Laboratory, University of Sussex, Brighton, East Sussex BN1 9QG, England, UK.
Tadayyon
Mohammad
M
Green
Irene C
IC
D002478:Cells, Cultured; D003470:Culture Media; D018076:DNA, Complementary; D015870:Gene Expression; D005947:Glucose / Q000008:administration & dosage; D006801:Humans; D007515:Islets of Langerhans / Q000378:metabolism*; D020411:Oligonucleotide Array Sequence Analysis; D015203:Reproducibility of Results
D003470:Culture Media; D018076:DNA, Complementary; D005947:Glucose