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 6 arrays of a 12-plex microarray slide which allowed us to hybridize 12 RNA samples: these corresponded to 6 pools of foraging and 6 pools of non-foraging workers from 6 different colonies.
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.
Monogyne colonies of Solenopsis invicta were collected near Athens (Georgia, USA) in April 2008. These colonies were kept at room temperature and fed standard ant diet which is composed of macerated laboratory-reared insects, pureed beef, eggs, and vitamins in agar for three months. At the beginning of the experiment, a plaster nest containing the colony (brood, workers and the queen) was put into a plastic box. The box was separated from a second plastic box (the foraging area) containing a frozen cricket, sugar water and water tubes. Food and water were supplied twice a week at regular days in the foraging area only. Ants needed to cross from the nest area to the foraging area through a plastic tube 100 cm long to get access to the food. The top of the rearing boxes and the external face of the tubes were covered with Teflon to prevent ants from escaping.
We assigned workers to two treatment groups according to the observed behavior at the moment of collection and independently of the age or size. We labeled as “foraging workers” (out) all the individuals that were collected in the foraging area in the process of handling food, water or sugar water, while we labeled as “non-foraging workers” (in) all the ants that were sampled within the nesting chamber in close proximity to the brood pile. Samples collected from both experiments were 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).