Selection of a suitable internal control gene for expression studies in pancreatic islet grafts.
The use of real-time reverse transcription polymerase chain reaction to compare gene expression in different tissues and conditions requires normalization to an internal control that must be expressed at a constant level. Although a previous validation step is required to confirm that an internal control is appropriate, no comparison of frequently used "housekeeping" genes is available for islet grafts. We have investigated the effect of transplantation and metabolic environment on the expression of 18S, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), beta-actin, and cyclophilin A genes in pancreatic islets. The expression of these genes was determined on days 1, 3, and 7 after transplantation into normoglycemic or hyperglycemic rats and in isolated islets. Only 18S gene expression remained stable in all studied conditions, indicating that it is the best internal control for gene expression analysis in islet grafts. The significant variation found in other housekeeping genes, particularly GAPDH and beta-actin, question their use as internal controls in islet grafts.
Rodriguez-Mulero
Sílvia
S
Laboratory of Diabetes and Experimental Endocrinology, Department of Medicine, IDIBELL-University of Barcelona, Spain.
Montanya
Eduard
E
D000199:Actins / Q000235:genetics; D000818:Animals; D001786:Blood Glucose; D021984:Cyclophilin A / Q000235:genetics; D003921:Diabetes Mellitus, Experimental / Q000235:genetics / Q000601:surgery*; D004195:Disease Models, Animal; D020869:Gene Expression Profiling / Q000592:standards*; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics; D006943:Hyperglycemia / Q000235:genetics / Q000601:surgery; D016381:Islets of Langerhans Transplantation; D008297:Male; D012337:RNA, Ribosomal, 18S / Q000235:genetics; D051381:Rats; D011917:Rats, Inbred Lew; D012015:Reference Standards; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000592:standards*
D000199:Actins; D001786:Blood Glucose; D012337:RNA, Ribosomal, 18S; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D021984:Cyclophilin A