Horm Metab Res 2006; 38(1): 8-11
DOI: 10.1055/s-2006-924968
Original Basic
© Georg Thieme Verlag KG Stuttgart · New York

Real-time PCR: Housekeeping Genes in the INS-1E β-cell Line

K.  Smidt1 , L.  Wogensen2 , B.  Brock1 , O.  Schmitz1, 3 , J.  Rungby1, 3
  • 1Department of Clinical Pharmacology, University of Aarhus, Denmark
  • 2Research Laboratory for Biochemical Pathology, Aarhus University Hospital, NBG, Denmark
  • 3Departments of Endocrinology and Metabolism C+M, Aarhus University Hospital, Aarhus, Denmark
Weitere Informationen

Publikationsverlauf

Received 23 June 2005

Accepted after revision 8 September 2005

Publikationsdatum:
13. Februar 2006 (online)

Abstract

Investigation of gene expression is a developing area with several methods available. One method is quantitative PCR. A major pitfall in quantitative PCR is the normalisation procedure of the gene expression. Many experiments include a housekeeping gene, some use RNA concentration, and others use a geometric mean of several internal, stably expressed genes. This study demonstrates that real-time-PCR results differ with varying housekeeping genes and analysis protocols when applied to insulin-secreting INS-1E cells derived from the pancreas and stimulated by DEDTC (diethyldithiocarbamate, a zinc chelator) and GLP-1.

References

  • 1 Santangelo C, Scipioni A, Marselli L, Marchetti P, Dotta F. Suppressor of cytokine signaling gene expression in human pancreatic islets: modulation by cytokines.  Eur J Endocrinol. 2005;  152 485-489
  • 2 Cardozo A K, Ortis F, Storling J, Feng Y M, Rasschaert J, Tonnesen M, Van Eylen F, Mandrup-Poulsen T, Herchuelz A, Eizirik D L. Cytokines downregulate the sarcoendoplasmic reticulum pump Ca2+ ATPase 2b and deplete endoplasmic reticulum Ca2+, leading to induction of endoplasmic reticulum stress in pancreatic beta-cells.  Diabetes. 2005;  54 452-461
  • 3 Brock B, Mogensen J H, Gregersen S, Hermansen K. Glucose desensitization in INS-1 cells: evidence of impaired function caused by glucose metabolite(s) rather than by the glucose molecule per se.  Metabolism. 2002;  51 671-677
  • 4 Yang J, Wong R K, Wang X, Moibi J, Hessner M J, Greene S, Wu J, Sukumvanich S, Wolf B A, Gao Z. Leucine culture reveals that ATP synthase functions as a fuel sensor in pancreatic beta-cells.  J Biol Chem. 2004;  279 53 915-53 923
  • 5 Diraison F, Parton L, Ferre P, Foufelle F, Briscoe C P, Leclerc I, Rutter G A. Over-expression of sterol-regulatory-element-binding protein-1c (SREBP1c) in rat pancreatic islets induces lipogenesis and decreases glucose-stimulated insulin release: modulation by 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR).  Biochem J. 2004;  378 769-778
  • 6 Nagai K, Tsuchiya K, Ezaki T, Tsuchiya M, Ohgawara H. Effect of GLP-1 (glucagon-like peptide 1 : 7 - 36 amide) on porcine pancreatic endocrine cell proliferation and insulin secretion.  Pancreas. 2004;  28 138-145
  • 7 Kola B, Korbonits M, Diaz-Cano S, Kaltsas G, Morris D G, Jordan S, Metherell L, Powell M, Czirjak S, Arnaldi G, Bustin S, Boscaro M, Mantero F, Grossman A B. Reduced expression of the growth hormone and type 1 insulin-like growth factor receptors in human somatotroph tumours and an analysis of possible mutations of the growth hormone receptor.  Clin Endocrinol (Oxf). 2003;  59 328-338
  • 8 Schmittgen T D, Zakrajsek B A. Effect of experimental treatment on housekeeping gene expression: validation by real-time, quantitative RT-PCR.  J Biochem Biophys Methods. 2000;  46 69-81
  • 9 Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, Speleman F. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes.  Genome Biol. 2002;  3 RESEARCH0034
  • 10 Palmiter R D, Cole T B, Quaife C J, Findley S D. ZnT-3, a putative transporter of zinc into synaptic vesicles.  Proc Natl Acad Sci USA. 1996;  93 14 934-14 939
  • 11 Cole T B, Wenzel H J, Kafer K E, Schwartzkroin P A, Palmiter R D. Elimination of zinc from synaptic vesicles in the intact mouse brain by disruption of the ZnT3 gene.  Proc Natl Acad Sci USA. 1999;  96 1716-1721
  • 12 Chimienti F, Devergnas S, Favier A, Seve M. Identification and Cloning of a β-Cell-Specific Zinc Transporter, ZnT-8, Localized Into Insulin Secretory Granules.  Diabetes. 2004;  53 2330-2337
  • 13 Weir G C, Mojsov S, Hendrick G K, Habener J F. Glucagonlike peptide I (7 - 37) actions on endocrine pancreas.  Diabetes. 1989;  38 338-342
  • 14 Buteau J, Roduit R, Susini S, Prentki M. Glucagon-like peptide-1 promotes DNA synthesis, activates phosphatidylinositol 3-kinase and increases transcription factor pancreatic and duodenal homeobox gene 1 (PDX-1) DNA binding activity in beta (INS-1)-cells.  Diabetologia. 1999;  42 856-864
  • 15 Lazaris J A, Babelskyi F J. Preventive effect of diethyldithiocarbamate on experimental diabetes produced by dithisone and 8-oxychinoline. Endocrinol.  Exp. 1979;  13 39-51
  • 16 Asfari M, Janjic D, Meda P, Li G, Halban P A, Wollheim C B. Establishment of 2-mercaptoethanol-dependent differentiated insulin-secreting cell lines.  Endocrinology. 1992;  130 167-178
  • 17 Merglen A, Theander S, Rubi B, Chaffard G, Wollheim C B, Maechler P. Glucose sensitivity and metabolism-secretion coupling studied during two-year continuous culture in INS-1E insulinoma cells.  Endocrinology. 2004;  145 667-678

Jørgen Rungby

Department of Clinical Pharmacology · University of Aarhus · Denmark ·

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