Gene expression analysis of calcineurin isoforms in T-lymphocytes--a method applied on kidney-transplant recipients.
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.


CONCLUSIONS
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.
Research Laboratory C, Department of Renal Medicine C, Aarhus University Hospital, Skejby, Brendstrupgaardsvej 100, DK 8200 Aarhus N, Denmark. dorthe.mortensen@farm.au.dk
D M
DM
Mortensen
R
R
Røge
A
A
Øzbay
P B
PB
Koefoed-Nielsen
K A
KA
Jørgensen
D000328:Adult; D019703:Calcineurin / Q000235:genetics / Q000378:metabolism; D005260:Female; D005500:Follow-Up Studies; D020869:Gene Expression Profiling; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D016030:Kidney Transplantation; D008297:Male; D008875:Middle Aged; D010744:Phosphoric Monoester Hydrolases / Q000235:genetics* / Q000378:metabolism*; D020033:Protein Isoforms / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013601:T-Lymphocytes / Q000378:metabolism*
D020033:Protein Isoforms; C120817:calcineurin phosphatase; D019703:Calcineurin; D010744:Phosphoric Monoester Hydrolases