Comment[ArrayExpressAccession] E-GEOD-48270 MAGE-TAB Version 1.1 Public Release Date 2013-09-01 Investigation Title Molecular Characterization of Heat Intolerance in Mice (microRNA data) Comment[Submitted Name] Molecular Characterization of Heat Intolerance in Mice (microRNA data) Experiment Description Prolonged exposure to high temperatures may cause heat-related illnesses, such as cramps, syncope, exhaustion or even stroke in some individuals. Heat-related injuries remain a threat to the health and operational effectiveness of military personnel, athletes and the general public. Heat injury victims experience long-term complications that may include multi-system organ (liver, kidney, muscle) and neurologic damage, as well as reduced exercise capacity and heat intolerance. Findings from our laboratory using a developed heat stress model show that about 1/3 of mice are heat-intolerant and vulnerable to heat injury even though they are from the same mice litter. We examined if there is any genetic causation to this pattern of observation between the two groups of mice classified (Heat Intolerant and Heat Tolerant). We would like to screen Heat Tolerant and Heat Intolerant mice samples using microarray technology and examine their microRNA and mRNA for possible gene-specific differences between the two groups (6 mice per group). The results from this proposed animal research will help identify and select potential markers that can be used as a pre-screen to identify heat intolerance and assess heat injury recovery in humans. Heat-induced physiological and biochemical changes were assessed to determine heat tolerance levels in mice. We performed mRNA and microRNA expression profiling on mouse gastrocnemius muscle tissue samples to determine novel biological pathways associated with heat tolerance. 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 Ghimbovschi Ghimbovschi Islam Person First Name Svetlana Svetlana Aminul Person Email geo@ncbi.nlm.nih.gov Person Affiliation Children's National Medical Center Person Address Research Center for Genetic Medicine, Children's National Medical Center, 111 Michigan Ave., NW, Washington, DC, USA Person Roles submitter Protocol Name P-GSE48270-1 P-GSE48270-5 P-GSE48270-6 P-GSE48270-2 P-GSE48270-3 P-GSE48270-4 P-GSE48270-7 Protocol Description AffymetrixM-BM-. miRNA QC Tool v 1.1.1.0 (Affymetrix, Santa Clara, CA) was used for the data summarization and microarrays quality control. Gene expression values were obtained using Affymetric Expression Console program, RMA-DABG option. ID_REF = VALUE = Quantification DETECTION P-VALUE = Total RNA samples, containing low molecular weight RNA, were biotin-labeled with FlashTagM-bM-^DM-" Biotin HSR RNA Labeling Kit (Genisphere LLC, Hatfield, PA). The quality of the biotin labeling process was confirmed by using Enzyme Linked Oligosorbent Assay (ELOSA) protocol (Genisphere LLC, Hatfield, PA). 21.5ul of each high-quality biotin-labeled miRNA sample was hybridized to Affymetrix Gene-ChipM-. miRNA 2.0 Array for 16 hours according to Affymetrix protocol. The arrays were washed and stained on the Affymetrix Fluidics station 400. Heat tests were conducted in an environmental chamber (Model 3950, Thermo Forma, Marietta, OH). Mice were placed in the chamber at ~21M-0C (relative humidity: ~22-30%) a day before experimentation. Heat exposures began the following morning after stable baseline data were obtained. Food and water were removed from cages before exposure. We used a modifiedheat protocol from the laboratory of Michel et al. (2007) such that heat exposure continued a maximum of 3 h, including ,1 h of heating the chamber to a predetermined temperature (39.5 ^ 0.28C). Heat exposure was terminated at core temperature (Tc) of ,42.48C or 3 h into heat exposure, whichever happened first. Based on our preliminary data, this protocol allowed sufficient heat exposure to test sensitivity to heat stress without any risk to the animals. All heat tests and telemetry measurements made to identify TOL and INT mice were performed after. All mice were maintained in conventional animal facilities (~21M-0C) with ad libitum food and water at the Uniformed Services University (USU) Laboratory Animal Medicine facility. The USU Institutional Animal Care and Use Committee approved all procedures performed on animals. All experimental mice were surgically implanted with a temperature transponder (Model G2 E-Mitter, Mini Mitter Corp, Bend, OR). At least two weeks were allowed for recovery. At the time of the experimental protocols, all mice were healthy as evidenced by body weight gains (M-bM-^IM-% presurgical levels), normal behavior and no sign of infection. Mouse muscle tissue samples were subjected to total RNA isolation procedure using mirVanaM-bM-^DM-" miRNA Isolation Kit (Applied Biosystems/Ambion, Austin, TX). Concentration of each RNA sample was determined by NanoDropM-. spectrophotometer ND-1000 (NanoDrop Technologies, Wilmington, DE). The quality of RNA samples was assessed with Agilent 2100 Bioanalyzer (Agilent Technologies Inc., Santa Clara, CA). The arrays were scanned with a Hewlett Packard G2500A gene Array Scanner. Protocol Type normalization data transformation protocol labelling protocol hybridization protocol sample treatment protocol growth protocol nucleic acid extraction protocol array scanning protocol Comment[SecondaryAccession] GSE48270 Comment[GEOReleaseDate] 2013-09-01 Comment[ArrayExpressSubmissionDate] 2013-06-25 Comment[GEOLastUpdateDate] 2013-09-03 Comment[AEExperimentType] transcription profiling by array SDRF File E-GEOD-48270.sdrf.txt