Hybridization parameters were determined for the reaction of complex cDNA probes to cDNA libraries carried on six nylon filters, each containing duplicate spots from 18,432 bacterial clones (macroarrays).
Lysate samples were printed at four serial dilutions (start concentration 0.3 mg/mL, plus 1.6-fold dilutions), each dilution as duplicate spots (in total eight spots per sample).
Signal intensities were log2 transformed and duplicate spots were averaged.
Each chip contained duplicate spots for every gene providing a means for basic statistical analysis.
A correlation was determined for the signal from duplicate spots of each probe.
Duplicate spots were located distally from each other to account for any intra-hybridization variation.
Duplicate spots were not averaged but treated as separate genes for analysis.
Signal intensities of duplicate spots were recorded and averaged for each antigen and for each cat serum, individually.
Duplicate spots within arrays and across technical replicate hybridisations (i.e. all replicate values for the same RNA sample) were averaged before performing the linear model fit.
Duplicate spots were averaged and filtered so that only genes for which there was a value for at least 4 replicates were statistically analysed.
As expected, duplicate spots were highly correlative with a total R squared of 0.99, similar to our previous published results from a Chlamydia trachomatis protein microarray [32].
