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Quantitative reverse transcription polymerase chain reaction shows that ethanol enhances interleukin-6 mRNA
1Department of Pathology and Laboratory Medicine, Tulane University, School of Medicine, New Orleans, LA 70112, USA.
Abstract:
IgA nephropathy, a form of mesangial glomerulonephritis, is associated with chronic ethanol ingestion in humans and in a rat model. We investigated the hypothesis that ethanol has a direct effect on a mesangial cell cytokine, interleukin-6 (IL-6). We measured IL-6 mRNA in cultured mesangial cells using a novel method, quantitative reverse transcription-polymerase chain reaction (Q-RT-PCR). Primers were used to amplify a 346-bp segment. To quantify the results, we generated an internal standard using site-directed mutagenesis which resulted in a restriction site for EcoRI, absent in target IL-6 cDNA. In the presence of the same primers, the mutated internal standard (exogenous template) amplifies with equal efficiency as the target IL-6 cDNA. Q-RT-PCR, using 250 ng of total RNA, showed that after ethanol incubation, IL-6 mRNA was 65 (+/-30) attomol, a 1.5-fold increase from 44 (+/-28) attomol in control cells. This study shows that in vitro, ethanol enhances IL-6 mRNA expression in rat mesangial cells.