Rodriguez-Mulero Sílvia, Montanya Eduard
Laboratory of Diabetes and Experimental Endocrinology, Department of Medicine, IDIBELL-University of Barcelona, Spain.
Transplantation. 2005 Sep 15;80(5):650-2. doi: 10.1097/01.tp.0000173790.12227.7b.
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.
使用实时逆转录聚合酶链反应来比较不同组织和条件下的基因表达，需要将其标准化至一个必须以恒定水平表达的内参基因。虽然之前需要一个验证步骤来确认一个内参基因是否合适，但目前尚无针对胰岛移植中常用“管家”基因的比较研究。我们研究了移植和代谢环境对胰腺胰岛中18S、甘油醛-3-磷酸脱氢酶（GAPDH）、β-肌动蛋白和亲环素A基因表达的影响。在移植到血糖正常或高血糖大鼠体内以及分离的胰岛中，于移植后第1、3和7天测定这些基因的表达。在所有研究条件下，只有18S基因表达保持稳定，这表明它是胰岛移植中基因表达分析的最佳内参基因。在其他管家基因中发现的显著差异，尤其是GAPDH和β-肌动蛋白，质疑了它们作为胰岛移植内参基因的适用性。
