Study of the relation between the activation of the inflammation in the encephalic death donor and the quality of the graft (time before the first rejection occurred). Diagnostic approach based on the profiling of the donor's inflammatory reaction. Keywords: procalcitonin, graft, inflammation
Overall design
The 22 patients of this study were divided in 2 groups corresponding to the extreme quartiles of PCT values. The first group are the patients with low PCT and the second group are the patients with high PCT. Blood sample: 2.5 mL harvested in PAXgene® Blood RNA tubes (PreAnalytix) RNA extraction: PAXgene® Blood RNA kit (Qiagen) We used a Universel Reference RNA (Stratagene) RNA amplification and labelling: kit Amino Allyl MessageAmp II (Ambion) We hybridized 4 microarrays per patient using pangenomic microarrays from the "Réseau National des Génopôles" (Illkirch, France). 2 slides were hybridized with reference RNA labelled Cy3 and patient RNA labelled Cy5, and 2 slides were hybridized with reference RNA labelled Cy5 and patient RNA labelled Cy3. Hybridation : Agilent protocol with few modifications : 750 ng of each labelled RNA were hubriddized at 60°C during 17 hours in an Aglient hybridization oven. After washings, Slides were scanned with a GenePix 4000B scanner (Molecular Devices). Image intensity data were extracted with GenePix Pro 6.0 analysis software. Quantification of Cy3 and Cy5 and selection of good spots were performed using the MAIA software (Novikov E and Barillot E. Software package for automatic microarray image analysis (MAIA). Bioinformatics. 2007 Mar 1;23(5):639-40. The ACUITY software was then used to normalize log ratios Cy3/Cy5 with Lowess non linear normalization, to filter out genes not present in at least 3 slides out of 4, to evaluate the reproducibility of the 4 microarrays of each patient (hierarchical clustering, Self Organizing Maps). Statistical analyses to insure reproducibility was performed using Excel (correlation coefficients, ANOVA). Only slides that passed all reprocubility tests were validated.
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