cDNA was resuspended in freshly prepared sodium carbonate buffer and dye coupled as per Whitfield et al 2003 [ 6], with the exception that dye-coupled samples were pooled only after purification on the Qiaquick PCR purification columns.
Here, the sampling ratios of all coupled sampling approaches were set to P 2 : P 1 = 0. 5.
The dye coupled sample and reference were stored at 4°C in the dark until hybridization.
We determined selenium concentrations in whole blood by inductively coupled plasma MS. Samples were diluted in ammonium hydroxide and then analyzed on an Elan 6000 instrument (Perkin Elmer-Sciex, Ontario, Canada).
Since only a couple of samples were positive for dihydrocodeine and 7-aminoflunitrazepam, these results are not plotted.
To avoid averaging out the F F dipolar coupling, the samples were run in a static condition.
The aa-dUTP-labeled cDNAs from sheared and static control samples were coupled to NHS-Cy5 and NHS-Cy3 (GE Healthcare), respectively.
The samples were coupled for 120 minutes and cleaned using a Cyscribe GFX purification kit (Amersham Bioscience, Little Chalfont, UK) according to the original instructions.
Following reverse transcription and clean up (alkaline hydrolysis and Qiaquick columns, Qiagen), cDNA samples were coupled to Alexa Fluor dyes (555, 647), using amino-allyl labeling methods and alternating direction with replicate arrays (i.e., dye-swap; [ 90]).
Each Cy3- (or Cy5-) coupled PAM sample was then hybridized to its corresponding Cy5- (or Cy3-) coupled reference sample anchored on a pig microarray slide containing 20,400 oligonucleotides (US Pig Genome Coordination Program; http://www.animalgenome.org/pig/resources/array_request.html).html
