title: Unstable Expression of Commonly Used Reference Genes in Rat Pancreatic Islets Early after Isolation Affects Results of Gene Expression Studies.
abstract: The use of RT-qPCR provides a powerful tool for gene expression studies; however, the proper interpretation of the obtained data is crucially dependent on accurate normalization based on stable reference genes. Recently, strong evidence has been shown indicating that the expression of many commonly used reference genes may vary significantly due to diverse experimental conditions. The isolation of pancreatic islets is a complicated procedure which creates severe mechanical and metabolic stress leading possibly to cellular damage and alteration of gene expression. Despite of this, freshly isolated islets frequently serve as a control in various gene expression and intervention studies. The aim of our study was to determine expression of 16 candidate reference genes and one gene of interest (F3) in isolated rat pancreatic islets during short-term cultivation in order to find a suitable endogenous control for gene expression studies. We compared the expression stability of the most commonly used reference genes and evaluated the reliability of relative and absolute quantification using RT-qPCR during 0-120 hrs after isolation. In freshly isolated islets, the expression of all tested genes was markedly depressed and it increased several times throughout the first 48 hrs of cultivation. We observed significant variability among samples at 0 and 24 hrs but substantial stabilization from 48 hrs onwards. During the first 48 hrs, relative quantification failed to reflect the real changes in respective mRNA concentrations while in the interval 48-120 hrs, the relative expression generally paralleled the results determined by absolute quantification. Thus, our data call into question the suitability of relative quantification for gene expression analysis in pancreatic islets during the first 48 hrs of cultivation, as the results may be significantly affected by unstable expression of reference genes. However, this method could provide reliable information from 48 hrs onwards.
authors.affiliation: Laboratory of Pancreatic Islets, Center of Experimental Medicine, Institute for Clinical and Experimental Medicine, Prague, Czech Republic.|First Faculty of Medicine, Charles University in Prague, Prague, Czech Republic.
authors.forename: Lucie
authors.initials: L
authors.lastname: Kosinová
authors.affiliation: Department of Metabolism and Diabetes, Center of Experimental Medicine, Institute for Clinical and Experimental Medicine, Prague, Czech Republic.
authors.forename: Monika
authors.initials: M
authors.lastname: Cahová
authors.affiliation: Laboratory of Pancreatic Islets, Center of Experimental Medicine, Institute for Clinical and Experimental Medicine, Prague, Czech Republic.
authors.forename: Eva
authors.initials: E
authors.lastname: Fábryová
authors.affiliation: Transplant Laboratory, Center of Experimental Medicine, Institute for Clinical and Experimental Medicine, Prague, Czech Republic.
authors.forename: Irena
authors.initials: I
authors.lastname: Týcová
authors.affiliation: Laboratory of Pancreatic Islets, Center of Experimental Medicine, Institute for Clinical and Experimental Medicine, Prague, Czech Republic.
authors.forename: Tomáš
authors.initials: T
authors.lastname: Koblas
authors.affiliation: Laboratory of Pancreatic Islets, Center of Experimental Medicine, Institute for Clinical and Experimental Medicine, Prague, Czech Republic.
authors.forename: Ivan
authors.initials: I
authors.lastname: Leontovyč
authors.affiliation: Laboratory of Pancreatic Islets, Center of Experimental Medicine, Institute for Clinical and Experimental Medicine, Prague, Czech Republic.|Department of Diabetes, Center of Diabetes, Institute for Clinical and Experimental Medicine, Prague, Czech Republic.
authors.forename: František
authors.initials: F
authors.lastname: Saudek
authors.affiliation: Department of Diabetes, Center of Diabetes, Institute for Clinical and Experimental Medicine, Prague, Czech Republic.
authors.forename: Jan
authors.initials: J
authors.lastname: Kříž
mesh_terms: D000818:Animals; D020869:Gene Expression Profiling; D007328:Insulin / Q000378:metabolism; D000078790:Insulin Secretion; D007515:Islets of Langerhans / Q000378:metabolism*; D008297:Male; D051381:Rats
chemical_list: D007328:Insulin