Investigation Title Transcription profiling of mouse neuron-specific mRNA complexity responses during hippocampal apoptosis following traumatic brain injury Comment[Submitted Name] RAD.Study[study_id=369]: Neuron specific mRNA complexity responses to Trauma Induced Apoptosis Experimental Design organism_part_comparison_design injury_design dye_swap_design transcription profiling by array Experimental Design Term Source REF mo mo mo EFO Comment[ArrayExpressReleaseDate] 2005-11-02 Comment[AEMIAMESCORE] 3 Comment[ArrayExpressAccession] E-CBIL-4 Comment[MAGETAB TimeStamp_Version] 2011-06-28 12:46:08 Last Changed Rev: 14857 Experimental Factor Name region conditions compared Experimental Factor Type organism_part disease_staging Experimental Factor Term Source REF Person Last Name McIntosh Person First Name Tracy Person Mid Initials Person Email mcintosh@seas.upenn.edu Person Phone 215-573-3156 Person Fax 215-573-3808 Person Address Department of Neurosurgery; 105 Hayden Hall, 3320 Smith Walk; Philadelphia; PA; 19104-6316; USA Person Affiliation University of Pennsylvania Person Roles submitter Person Roles Term Source REF Quality Control Type Quality Control Term Source REF Replicate Type Replicate Term Source REF Normalization Type Normalization Term Source REF Date of Experiment Public Release Date 2005-11-02 PubMed ID 15044525 Publication DOI 15044525 Publication Author List Marciano P.G., Brettschneider J., Manduchi E., Davies J.E., Eastman S., Raghupathi R., Saatman K.E., Speed T.P., Stoeckert C.J. Jr., Eberwine J.H., McIntosh T.K. Publication Title Neuron-specific mRNA complexity responses during hippocampal apoptosis following traumatic brain injury Publication Status journal_article Publication Status Term Source REF Experiment Description Study aimed at examining differential expression between single apoptotic neurons from both the CA-3 and dentate and those in an un-injured brain. Protocol Name cbil.upenn.edu:RAD.Protocol:984:Protocol cbil.upenn.edu:RAD.Protocol:1005:Protocol cbil.upenn.edu:RAD.Protocol:1007:Protocol cbil.upenn.edu:RAD.Protocol:1006:Protocol cbil.upenn.edu:RAD.Protocol:1008:Protocol cbil.upenn.edu:RAD.Protocol:505:Protocol Protocol Type specified_biomaterial_action dissect pool linear_amplification labeling hybridization Protocol Description Mouse is anesthetized with intraperitoneal injection of sodium pentobarbital (65 mg/kg), surgically prepared and subjected to CCI brain injury using a velocity of 5.0 m/sec and a depth of 1.0 mm. Throughout the surgery and during recovery, mouse is kept on a heating pad to maintain physiological body temperature. Following TUNEL and active caspase 3 immunohistochemistry, sections were incubated overnight with oligo-dT-T7. Transcription of the first strand of cDNA was performed in situ to increase the yield of cDNA from the endogenous mRNA pool prior to microdissection. After this, the sections were washed in 0.5X SSC and individual labeled cells were viewed on an inverted Olympus microscope. With the use of a RNAse free glass micropipette attached to a Narishige micromanipulator, individual neurons were carefully microdissected from the surrounding neuropil taking care not to disrupt any adjacent cells and subsequently aspirated into the micropipette for amplification. The microdissected neurons were either unstained, active caspase 3(+)/TUNEL(-), or active caspace 3(+)/TUNEL(+) from both the pyramidal layer of the CA-3 region and granule layer of the dentate gyrus. Digital photomicrographs in both fluorescence and bright fields were taken before and after aspiration to assure that more than 80% of the cell was dissected and that only a single cell was detached. If any part of an adjoining cell was perturbed or detached in the process the dissected cell was not included in further analyses. The double-stranded DNA products from phenotypically identical cells were pooled to decrease variability attained from non-littermates and from cell-to-cell variation. Each pooled probe was composed of a total of 6 cells from 3 different mice: one cell from each of two non-consecutive sections per animal. (The mRNA from individual neurons was amplified according to previously described methods by Eberwine et al.). Approximately 75% of the ds-DNA material was used for first round aRNA amplification with T7 RNA polymerase and the remaining 25% was kept for the Real-Time PCR validation. Radiolabeled CTP was used to visualize the probe on an RNA denaturing gel. Following the estimated 1000-2000 fold amplification, the newly synthesized aRNA was primed with random hexanucleotide primers and served as a template for cDNA synthesis. The labeled probe was synthesized by converting the amplified aRNA to either a Cy3 or a Cy5-labeled cDNA probe using a custom labeling kit as described by Yue et al., 2001. The fluorescently labeled cDNA was synthesized with Cy3 or Cy5 random 9mer (Trilink, CA) and MMLV RNase H-free reverse transcriptase (Promega, WI). Hybridization of labeled cDNA probes was performed in 20 ul of 5X SSC, 0.1% SDS, 1 mM DTT at 60 C for 6 hrs. Protocol Parameters Protocol Hardware Protocol Software Protocol Contact Protocol Term Source REF mo mo mo mo The MGED Ontology The MGED Ontology SDRF File E-CBIL-4.sdrf.txt Term Source Name NCI_thesaurus ncbitax The MGED Ontology emap_ts cbil_cv mo ArrayExpress mo EFO The MGED Ontology Term Source File ncithesaurus.obo.alt http://www.ncbi.nlm.nih.gov/Taxonomy/taxonomyhome.html http://mged.sourceforge.net/ontologies/MGEDontology.php http://genex.hgu.mrc.ac.uk/Databases/Anatomy/Diagrams/ http://www.cbil.upenn.edu/anatomy.php3 http://mged.sourceforge.net/ontologies/MGEDontology.php http://www.ebi.ac.uk/arrayexpress http://mged.sourceforge.net/ontologies/MGEDontology.php http://www.ebi.ac.uk/efo/ http://mged.sourceforge.net/ontologies/MGEDontology.php Term Source Version