Subscribe to RSS
DOI: 10.1055/s-2006-924968
Real-time PCR: Housekeeping Genes in the INS-1E β-cell Line
Publication History
Received 23 June 2005
Accepted after revision 8 September 2005
Publication Date:
13 February 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.
Key words
DEDTC - GLP-1 - UBC-7 - HPRT - cyclophilin A - ZnT-3 - ZnT-8
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 ·
Phone: +45 8942 1708
Fax: +45 8612 8804
Email: jr@farm.au.dk