Array development
To generate the genetic arrays, cDNA clones were isolated from four subtractive libraries derived from worker- and queen-destined larvae collected in the third and fourth instars. We chose 144 clones from worker-biased libraries and 144 clones from queen-biased libraries. These clones were amplified by the polymerase chain reaction (PCR) using endogenous adaptor primers (primers Nest1 and Nest2r, described in [3]), then separated and verified by agarose gel electrophoresis. After being denatured for 5 min at 95°C in a final molarity 0.2 M NaOH, approximately 5 μg DNA from each amplified clone was fixed onto nylon membranes (Hybond N+, Amersham) using a 5 μl slot-pin library replicator (V and P Scientific) followed by UV irradiation. Each of 96 quadrants in a 384-cell printing contained three samples and a negative control. An aliquot of each cDNA clone was purified then sequenced using fluorescent-dye labeling and an ABI Prism 373 DNA analysis machine (Applied Biosystems).