Validation of internal control genes for gene expression analysis in diabetic glomerulosclerosis

Abstract

Background. Gene expression analysis is an invaluable tool in the study of diabetic glomerulosclerosis. The necessary denominator for the quantitative expression of a specific gene is the expression level of a second gene that is presumed to remain unchanged. Thus, it is critical that the stability of this housekeeping gene in diabetic glomeruli or in cultured glomerular cells is not altered by the disease or a high glucose environment, respectively. Although gene expression quantification, achieved by Northern blot analysis or real-time reverse transcription-polymerase chain reaction (RT-PCR) has been extensively applied in diabetic renal tissue in vivo and in vitro, there are no studies validating the use of any specific endogenous control gene in these measurements. Methods. We performed real-time RT-PCR using RNA from microdissected diabetic glomeruli and from mesangial cells cultured in high glucose concentration to investigate gene expression stability of β-actin, glyceraldehyde-3-phosphate dehydrogenase (GADPH), phospholipase A2, β2-microglobulin, acidic ribosomal protein 36B4, and cyclophilin A. Results. Using an analysis method which is independent of gene abundance and compares the pair-wise variation of a given housekeeping gene with all other control genes, β-actin and phospholipase A2, were found to be the most stable genes in diabetic glomeruli and in primary mesangial cells exposed to 20 mmol/L glucose. Conclusion. It is proposed that the expression level of these genes is the best reference to evaluate relative changes in gene activity in diabetic/high glucose exposed glomerular tissues. © 2004 by the International Society of Nephrology.