The kinase reactions (1× PK kinase buffer, JAK proteins at indicated concentration, 1× BSA, 12.5 µg/ml fluorescein-labeled PY20 antibody and 400 µM ATP) were incubated for 60 cycles of pumping up and down through the pores of the microarrays at a rate of 2 cycles per minute.
Many of these methods were established during the early days of microarrays at a time when experimental procedures were still being optimized and technical variability was omnipresent.
Immunohistochemistry was carried out on tissue microarrays at a dilution of 1 : 1000 followed by detection with the En Vision+ System using the peroxidase method (DAKO, Carpinteria, CA, USA).
There were two transcripts, DLX5 and DCN, which were detected as differentially expressed by qPCR but not by the microarrays at a statistically conservative Bonferroni corrected p < 0.05 cutoff.
All 12 measured genes showed a similar expression tendency than when detected by microarray analysis, and 9 of them correlated with the results obtained using Affymetrix microarrays at a significance of P<0.05.
In Phase I, serum samples from 22 AIH patients and 30 healthy controls were selected and individually used to probe the human protein microarrays at a 1000-fold dilution, followed by detection of bound human autoantibodies using a Cy-5-conjugated anti-human IgG antibody.
