D M Mortensen1, R Røge, A Øzbay, P B Koefoed-Nielsen, K A Jørgensen. 1. Research Laboratory C, Department of Renal Medicine C, Aarhus University Hospital, Skejby, Brendstrupgaardsvej 100, DK 8200 Aarhus N, Denmark. dorthe.mortensen@farm.au.dk
Abstract
BACKGROUND: Tacrolimus exerts its immunosuppressive effect through inhibition of the intracellular enzyme calcineurin phosphatase (CaN). In this study, we set-up a validated real-time PCR method to measure the gene expression of the two major isoforms of the catalytic subunit of CaN in T-lymphocytes. METHODS: 20 stable kidney-transplant recipients, 10 early kidney-transplant recipients and 10 healthy non-medicated subjects had blood drawn and T-lymphocytes were isolated using E-rosette gradient centrifugation method. The cell counts were analyzed by DNA quantification using Hoeschst 33285. Gene expressions were analyzed using real-time PCR for CaN Aalpha, CaN Abeta and the reference genes CD3E and PPIB. RESULTS: The real-time PCR method was found to be with high efficiencies and low intra- and inter-assay variabilities. No statistically significant differences were found in the gene expression levels of the two reference genes among the three groups. The two major isoforms of CaN A were expressed in equal amounts in the T-lymphocytes. CONCLUSION: We found no significant difference in the reference genes between the three groups, but looking at the data there was a trend towards an up-regulation of CD3E. PPIB appears to be the more stable of the two reference genes tested in our study. Copyright (c) 2010 Elsevier B.V. All rights reserved.
BACKGROUND:Tacrolimus exerts its immunosuppressive effect through inhibition of the intracellular enzyme calcineurin phosphatase (CaN). In this study, we set-up a validated real-time PCR method to measure the gene expression of the two major isoforms of the catalytic subunit of CaN in T-lymphocytes. METHODS: 20 stable kidney-transplant recipients, 10 early kidney-transplant recipients and 10 healthy non-medicated subjects had blood drawn and T-lymphocytes were isolated using E-rosette gradient centrifugation method. The cell counts were analyzed by DNA quantification using Hoeschst 33285. Gene expressions were analyzed using real-time PCR for CaN Aalpha, CaN Abeta and the reference genes CD3E and PPIB. RESULTS: The real-time PCR method was found to be with high efficiencies and low intra- and inter-assay variabilities. No statistically significant differences were found in the gene expression levels of the two reference genes among the three groups. The two major isoforms of CaN A were expressed in equal amounts in the T-lymphocytes. CONCLUSION: We found no significant difference in the reference genes between the three groups, but looking at the data there was a trend towards an up-regulation of CD3E. PPIB appears to be the more stable of the two reference genes tested in our study. Copyright (c) 2010 Elsevier B.V. All rights reserved.
Authors: Madhura Modak; Otto Majdic; Petra Cejka; Sabrina Jutz; Alexander Puck; Jens G Gerwien; Peter Steinberger; Gerhard J Zlabinger; Herbert Strobl; Johannes Stöckl Journal: Immunology Date: 2016-08-16 Impact factor: 7.397
Authors: Alexander Puck; Stefan Hopf; Madhura Modak; Otto Majdic; Petra Cejka; Stephan Blüml; Klaus Schmetterer; Catharina Arnold-Schrauf; Jens G Gerwien; Klaus S Frederiksen; Elisabeth Thell; Judith Leitner; Peter Steinberger; Regina Aigner; Maria Seyerl-Jiresch; Gerhard J Zlabinger; Johannes Stöckl Journal: Eur J Immunol Date: 2016-10-31 Impact factor: 5.532