Comment[ArrayExpressAccession]	E-GEOD-24953
MAGE-TAB Version	1.1						
Public Release Date	2013-10-26
Investigation Title	Effects of mating on gene expression profiles of mature ant queens						
Comment[Submitted Name]	Effects of mating on gene expression profiles of mature ant queens
Experiment Description	Will be added/updated once the manuscript is finalized. Cardiocondyla obscurior queens. Three treatments: virgin queens, queens mated by real males, queens sham-mated (by sterile males). Queens collected 1 week and 8 week after mating. seven loops for queens collected 1 week after mating; nine loops for queens collected 8 weeks after mating. Five direct comparisons (with dye-swaps... so 10 arrays) were done of between 1 week and 8 week samples of queens mated by real males. Each sample is RNA from two queens (from different colonies). Samples were hybridized against Solenopsis invicta microarrays (signal was detectable for most clones!)						
Term Source Name	ArrayExpress	EFO
Term Source File	http://www.ebi.ac.uk/arrayexpress/	http://www.ebi.ac.uk/efo/efo.owl					
Person Last Name	Wurm	Oettler	Wang	Wurm	Keller	Heinze	
Person First Name	Yannick	Jan	John	Yannick	Laurent	JM-CM-<rgen	
Person Email	yannick.wurm@unil.ch						
Person Affiliation	University of Lausanne						
Person Address	Ecology and Evolution, University of Lausanne, Biophore, Lausanne, Switzerland						
Person Roles	submitter						
Protocol Name	P-GSE24953-1	P-GSE24953-5	P-GSE24953-6	P-GSE24953-2	P-GSE24953-3	P-GSE24953-4	P-GSE24953-7
Protocol Description	Spot intensities were quantified from scanned microarray images with GenePix Pro 6.0 using default settings and manual correction and flagging of ambiguous spots. Subsequently, we followed a standard microarray analysis procedure, guided by the documentation of the Bioconductor limma package (Gentleman et al. 2004; Smyth 2004; Smyth et al. 2005). In brief, median signal and background levels were extracted for each cDNA spot. The limma 3.2.1 package (Smyth 2004) in R 2.10.1 (The R Development Core Team 2007) was used for Normexp background correction, Print-tip Loess normalization within arrays, and Aquantile normalization between arrays (Smyth & Speed 2003). ID_REF =  VALUE = print tip loess normalized M value representing log2(cy5/cy3)	Total RNA was amplified using the Ambion Message Amp II kit. Amplified RNA plus Alien spike control RNAs (Stratagene) were labeled using the indirect aa-dUTP labeling method. Reverse transcription primers: random 9mers.	Hybridization buffer (3x SSC, 0.18% SDS, 200 ng/M-NM-<l yeast tRNA, 200 ng/M-5l polyA DNA; final concentration) was added to samples; samples were denatured, applied to microarrays, and hybridized overnight at 64M-0C. After hybridization, slides were washed sequentially.	We sampled one and eight week old queens that have been subjected to one of three mating treatments. We analyzed 7 and 9 biological replicates for each treatment (M, S and V) at the respective time point (one and eight week old queens). To reduce inter-individual variation within replicate, we pooled two queens per sample for each treatment at each time point. Thus, in total, we sampled 42 one-week-old and 54 eight-week-old queens resulting in a total of 96 experimental colonies.For the treatment with sterilized males we collected males one to two days after eclosion and exposed them, individually placed in 100ul cups, to x-ray (120 Gy; 2,95 M-1 0.12 Gy/min, ca 40min) which results in sperm degradation (Schrempf et al 2005), a dose that also induces sterility in similar-sized braconidae wasps (Grosch and Sullivan 1954). After irradiation we immediately returned the males to their colony. At this stage the queens were between 2 and 3 days old. Some males (~ 10%) died within the first few hours after irradiation, showing black cuticular pigmentation and spastic body movements. Other males were initially disorientation compared to non-treated males. Males that survived the first 24h appeared to recover and showed typical male behaviour, such as patrolling the nest. Observations of attempted mating confirmed the sexual functionality of these males. Colonies in which males (of both mated and dummy mated treatment) did not survive for more than 10 days were excluded to further ensure for the functionality of the males. After 10 days all males were removed. S and M queens shed their wings within days after mating and therefore we excluded the few cases where queens remained winged, indicating that something unexpected happened, i.e either the queens or their mating partners did not engage in full mating behavior.	Each colony was started with 20 adult workers, 10 worker pupae, brood (8 larvae of each of the three stages, cf. Schrempf & Heinze 2006) and a single not-yet sclerotized queen pupae (Fig. 1). Colonies were housed in petri-dishes (M-bM-^HM-^E = 10cm) with plaster-flooring in which small cavities have been carved to serve as nest sites. Colonies were fed three times a week with chopped cockroaches, Drosophila and diluted honey. We observed each colony of the 8 week old queens three times a week for developing male and queen pupae which we removed to avoid re-mating (although unlikely in Cardiocondyla) or mistaking a young queen for the original one. We provisioned the colonies with brood and worker pupae from random chosen stock donor colonies, when necessary. After one or eight weeks, respectively, queens were collected individually in 1.5ml microcentrifuge tubes, immediately snap-frozen in liquid N2 and stored at -80M-0C.	We extracted total RNA from whole queens with the Trizol Reagent (Invitrogen, Carlsbad, CA), following the manufacturers protocol. Tissue was homogenized with ceramic beads (Quackenbush) using a Fastprep bead shaker. Extraction of two queens resulted in a total RNA yield of 25 M-bM-^@M-^S 40M-5g after DNAse treatment (Ambion DNA-free, Austin, TX) and subsequent Microcon purification (Millipore, Billerica, MA). Extracted RNA was dried at 50M-0C in a vacuum centrifuge and re-dissolved in 12M-5l H2O. 10M-5l of total RNA was used for amplification (MessageAmp II aRNA Amplification Kit, Ambion, Austin, TX) yielding 110-1625M-5g of amplified RNA (aRNA) for each sample.	Scanner: Agilent Technologies microarray scanner. Image quantification GenePix6.0
Protocol Type	normalization data transformation protocol	labelling protocol	hybridization protocol	sample treatment protocol	growth protocol	nucleic acid extraction protocol	array scanning protocol
Experimental Factor Name	TIMESINCEMATING	MATINGSTATUS					
Experimental Factor Type	timesincemating	matingstatus					
Comment[SecondaryAccession]	GSE24953						
Comment[GEOReleaseDate]	2013-10-26						
Comment[ArrayExpressSubmissionDate]	2010-10-27						
Comment[GEOLastUpdateDate]	2013-10-27						
Comment[AEExperimentType]	transcription profiling by array						
SDRF File	E-GEOD-24953.sdrf.txt