Reproducibility of targeted gene expression measurements in human islets of Langerhans

R. Barry, M. Tadayyon, I.C. Green

Research output: Contribution to journalArticle

Abstract

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.
Original languageEnglish
Pages (from-to)350-356
Number of pages7
JournalBiochemical and biophysical research communications
Volume298
Issue number3
DOIs
Publication statusPublished - Nov 2002

Fingerprint

Islets of Langerhans
Gene Expression
Collodion
Oligonucleotide Array Sequence Analysis
Complementary DNA
Pancreas
RNA
Insulin
Apoptosis
Glucose
Messenger RNA
Membranes

Keywords

  • Human islets of Langerhans
  • Targeted gene expression
  • Glucotoxicity
  • Insulin secretion
  • Insulin synthesis
  • Oxidative stress
  • Radical scavenging
  • Cell signalling
  • Fas/FasL
  • Apoptosis

Cite this

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abstract = "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.",
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Reproducibility of targeted gene expression measurements in human islets of Langerhans. / Barry, R.; Tadayyon, M.; Green, I.C.

In: Biochemical and biophysical research communications, Vol. 298, No. 3, 11.2002, p. 350-356.

Research output: Contribution to journalArticle

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AU - Barry, R.

AU - Tadayyon, M.

AU - Green, I.C.

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AB - 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.

KW - Human islets of Langerhans

KW - Targeted gene expression

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KW - Insulin secretion

KW - Insulin synthesis

KW - Oxidative stress

KW - Radical scavenging

KW - Cell signalling

KW - Fas/FasL

KW - Apoptosis

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