Gene expression profiling of cultured human islet preparations.
The expression of functional and regulatory genes by islet cells is a key determinant for the success of islet transplantation. The aim of this study is twofold: first, to characterize the cluster of genes expressed in human islet isolations; and second, to validate the capability of gene array technology to assess with accuracy the expression of various transcripts. RNA from isolated islet preparations obtained from three independent donors was converted to cDNA and then transcribed to cRNA. Individual cRNA preparations were then hybridized to U133A microarrays carrying approximately 23,000 genes, and analyzed using GeneSpring (SiliconGenetics, Redwood City, CA) software. Real-time reverse transcription-polymerase chain reaction was performed to validate results obtained by microarray analysis. Microarray analysis identified the expression of about 7,000 genes transcribed in cultured human islet preparations. Enzymes represented the most abundant class of genes identified, followed by nuclear binding proteins, signal transduction molecules, transport proteins, and growth factor receptors and their ligands. Real-time polymerase chain reaction confirmed the identification of various islet-specific genes detected by microarray analysis, but also showed that such genes as pancreatic duodenal homeobox 1 protein and glucagon-like peptide 1 receptor, which were not detected by gene array, can be readily identified and quantified. In addition, gene array produced a suboptimal quantification of genes expressed in large amounts by islet cells. Indeed, the abundance of mRNA for insulin when compared with the level of somatostatin mRNA was not as different as one would have predicated based on the classic knowledge of islet physiology. Gene array analysis appears to be a valuable tool to obtain preliminary information of genes expressed by a given tissue. The expression levels of transcripts expressed in very low or very high quantities need to be confirmed by an independent technique.
Hui
Hongxiang
H
Division of Endocrinology, Diabetes, and Metabolism, Department of Medicine, Cedars-Sinai Medical Center, Los Angeles, California 90048, USA.
Wang
Charles
C
Li
Hongmei
H
Bulotta
Angela
A
D'Amico
Eugenio
E
Khoury
Nasif
N
Nguyen
Elizabeth
E
Di Mario
Umberto
U
Chen
Ida Y
IY
Perfetti
Riccardo
R
D001483:Base Sequence; D002478:Cells, Cultured; D017931:DNA Primers; D018076:DNA, Complementary / Q000235:genetics; D020869:Gene Expression Profiling; D006801:Humans; D007515:Islets of Langerhans / Q000502:physiology*; D009693:Nucleic Acid Hybridization; D020411:Oligonucleotide Array Sequence Analysis; D016133:Polymerase Chain Reaction; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D014158:Transcription, Genetic
D017931:DNA Primers; D018076:DNA, Complementary; D012313:RNA