Comment[ArrayExpressAccession]	E-GEOD-49136
MAGE-TAB Version	1.1						
Public Release Date	2013-07-24
Investigation Title	Toxicity and transcriptomic analysis in Hyalella azteca suggests increased exposure and susceptibility of epibenthic organisms to Zinc Oxide Nanoparticles						
Comment[Submitted Name]	Toxicity and transcriptomic analysis in Hyalella azteca suggests increased exposure and susceptibility of epibenthic organisms to Zinc Oxide Nanoparticles
Experiment Description	Abstract:  Nanoparticles (NPs) are expected to make their way into the aquatic environment where sedimentation of particles will likely occur, putting benthic organisms at particular risk.  Therefore, organisms such as Hyalella azteca, an epibenthic crustacean which forages at the sediment surface, is likely to have a high potential exposure.  Here we show that Zinc Oxide  (ZnO) NPs are more toxic to H. azteca compared with the corresponding metal ion, Zn2+.  Dissolution of ZnO NPs contributes about 50% of the Zn measured in the ZnO NP suspensions, and cannot account for the toxicity of these particles to H. azteca.  However, gene expression analysis is unable to distinguish between the ZnO NP exposures and Zinc Sulfate (ZnSO4) exposures at equitoxic concentrations.  These results lead us to hypothesize that ZnO NPs provide and an enhanced exposure route for Zn2+ uptake into H. azteca, and possibly other sediment dwelling organisms.  Our study supports the prediction that sediment dwelling organisms are highly susceptible to the effects of ZnO NPs and should be considered in the risk assessment of these nanomaterials. This experiment included four different treatments and an untreated control.  Each treatment or control, consisted of ten independent replicates of twenty Hyalella azteca.  Of these, six were randomly chosen to be used for the microarray analysis.						
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	Poynton	Poynton	Lazorchak	Blalock			
Person First Name	Helen	Helen	James	Bonnie			
Person Mid Initials	C	C	M				
Person Email	helen.poynton@umb.edu						
Person Affiliation	UMass Boston						
Person Phone	617-287-7323						
Person Address	Environmental, Earth and Ocean Sciences, UMass Boston, 100 Morrissey Blvd., Boston, MA, USA						
Person Roles	submitter						
Protocol Name	P-GSE49136-1	P-GSE49136-5	P-GSE49136-6	P-GSE49136-2	P-GSE49136-3	P-GSE49136-4	P-GSE49136-7
Protocol Description	Feature extraction was performed using Nimblescan software (Roche Nimblegen) and intensity values were normalized by quantile normalization with median polish and log2 transformed in Arraystar (DNAstar, Madison, Wisconsin). Sequences which were differentially expressed compared to the untreated control or between treatment groups were determined using Statistical Analysis for Microarrays (SAM)24 with a false positive cut-off of 1. All data normalization and statistical testing were performed at the probe level since a de novo trancriptome assembly of Hyalella azteca was being used. ID_REF =  VALUE = probe-level normalized values	RNA was reverse transcribed and labeled using Nimblegen one-color DNA labeling kit (Roche Nimblegen, Madison, WI) following the manufacturer’s protocols. RNA was amplified first using TransPlex WTA I Amplification kit (Sigma Aldrich) according to Nimblegen Technical note (Skalitsky and Watt, available at www.nimblegen.com/arraysupport).	Labeled cDNA was hybridized overnight to the custom 133k H. azteca arrays (described in Supporting Information) in a Nimblegen hybridization system following the manufacture’s protocols. Following hybridization, microarrays were washed with the Nimblegen wash buffer kit (Roche Nimblegen, Madison, WI) according to the manufacturer’s recommendations.	96-h static, water only exposures were performed as described above for the toxicity bioassays. Fresh 150 ml of R-MHRW containing the effective concentrations determined through toxicity testing , of metal-based NP or salt was added to each beaker with ten replicate beakers for each exposure concentration (n=6 for microarray analysis, n=4 for RT-qPCR). Twenty H. azteca, aged 10-days were added to each test beaker with a pipette. Test vessels were placed in an environmental chamber at 23oC for 96-h. The media was not agitated by bubbling or stirring during the 96-h exposure to better mimic the natural environment where H. azteca may be exposed to NPs.	H. azteca were maintained at the US EPA aquatic culturing facility in Cincinnati, OH according to standard methods: U.S. EPA. Methods for Measuring the Toxicity and Bioaccumulation of Sediment-associated Contaminants with Freshwater Invertebrates; EPA 600/R-99/064. U.S. ; U.S. Environmental Protection Agency, Office Research and Development: Duluth, Minnesota, 2000.	H. azteca were removed from exposure vessels with a pipette and immersed immediately in 0.75 ml of TriReagent (Molecular Research Center, Cincinnati, OH) in a 1.5 ml Eppendorf tube. All organisms in a single treatment vessel (20 animals) were pooled into one microcentrifuge tube. H. azteca tissue was homogenized for 5 min. at 30 vibrations/s using a Tissue Lyser bead mill (Qiagen Inc., Valencia, CA) with 3.2 mm stainless steel beads (Biospec Products, Bartlesville, OK) and RNA was isolated from the homogenate according to the TriReagent's manufacturer’s methods. RNA was precipitated on a RNAeasy spin column and treated on the column with DNAse I (Qiagen). Following DNAse treatment, RNA was eluted from with spin column with RNAse free water. RNA concentration and quality was assessed using a NanoDrop Spectrophotometer and by visual analysis of RNA on a 1.5 % agarose gel using NorthernMax-Gly buffer (Applied Biosystems/Ambion, Austin, TX).	DNA microarrays were scanned at 3 micron resolution using an Agilent DNA Microarray Scanner G2565CA (Agilent Technologies, Santa Clara, CA) at the University of Massachusetts Medical School in Worchester, MA. One of the six microarrays performed on the ZnO NP LC25 exposure was removed from down-stream analyses due to technical problems during the hybridization step, resulting in n=5 for this treatment group.
Protocol Type	normalization data transformation protocol	labelling protocol	hybridization protocol	sample treatment protocol	growth protocol	nucleic acid extraction protocol	array scanning protocol
Comment[SecondaryAccession]	GSE49136						
Comment[GEOReleaseDate]	2013-07-24						
Comment[ArrayExpressSubmissionDate]	2013-07-23						
Comment[GEOLastUpdateDate]	2013-07-24						
Comment[AEExperimentType]	transcription profiling by array						
SDRF File	E-GEOD-49136.sdrf.txt