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.
Mortensen
D M
DM
Research Laboratory C, Department of Renal Medicine C, Aarhus University Hospital, Skejby, Brendstrupgaardsvej 100, DK 8200 Aarhus N, Denmark. dorthe.mortensen@farm.au.dk
Røge
R
R
Øzbay
A
A
Koefoed-Nielsen
P B
PB
Jørgensen
K A
KA
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