normalization data transformation protocol
Background correction (with parameter “normexp+offset=50”) and normalization (“NormalizedWithinArrays” with parameter “loess” followed by “NormalizedBetweenArrays” method = “Aquantile”) were done in the “limma” package for R.
array scanning protocol
Arrays were scanned using Axon GenePix 4000B.
nucleic acid hybridization to array protocol
Samples were hybridized with mixing in a MAUI hybridization instrument overnight at 42 °C. We used all 12 arrays of a 12-plex microarray slide to hybridize 24 RNA samples from 6 different colonies, and for each colony we processed 1 pool of QRin, 1 of QLin, 1 of QRout and 1 of QLout.
nucleic acid labeling protocol
Fifteen µg of aRNA was labeled with Cy3 or Cy5 (GE Health Care, Pittsburgh, PA) and subsequently purified according to the Ambion Kit instructions. 1.5 µg of a Cy3 labeled sample were combined with 1.5 µg of a Cy5 labeled sample and fragmented using RNA Fragmentation Reagents (Ambion AM8740, Austin, TX) according to the manufacturer’s instructions.
After 5 days, we assigned workers to 4 treatment groups according to the observed behavior at the moment of collection and independently of the age or size. We labeled them as queenright non-foraging (QRin), queenless non-foraging (QLin), queenright foraging (QRout) and queenless foraging (QLout). Samples were collected between 9am and 1pm, flash frozen in dry ice and stored at -80 °C until processed for molecular work.
nucleic acid extraction protocol
Total RNA was extracted from pools of 10 worker ants (whole bodies) using the PicoPure RNA Isolation kit (Applied Biosystems – Life Technologies, Grand Island, NY) combined with an RNase-Free DNase step (Qiagen, Valencia, CA) to remove any possible contamination by genomic DNA. Concentration and purity of RNA samples were assessed using NanoDrop (Thermo Scientific, Wilmington, DE) and Qubit 2.0 Fluorometer (Life Technologies, Grand Island, NY) and RNA quality was assessed using RNA Nano Chips on the Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA). One µg of each sample was amplified using the Amino Allyl MessageAmp II aRNA Amplification Kit (AM1753, Ambion, Austin, TX).
Monogyne colonies of Solenopsis invicta were collected in the surroundings of Gainesville (Florida, USA) in April-May 2010. From each of 10 colonies we obtained two sub-colonies, one queenright (QR) and the other queenless (QL). Each sub-colony was housed in a plastic pencil box containing a small nesting chamber on one side, and a small foraging area on the other side with a frozen cricket, water and sugar water. In all colony fragments we transferred equal amounts of brood (a full 0.5ml plastic tube), workers (5ml) and winged females (virgin queens, ca. 10) from the original colony.