Investigation Title Transcription profiling of microdissected global and orbital layers of the rectus extraocular muscle (EOM) from adult Sprague-Dawley rats Comment[Submitted Name] Laminar gene expression of rat extraocular orbital and global layers Experimental Design organism_part_comparison_design co-expression_design transcription profiling by array Experimental Design Term Source REF mo mo EFO Comment[ArrayExpressReleaseDate] 2007-10-31 Comment[SecondaryAccession] GSE1374 Comment[AEMIAMESCORE] 4 Comment[SecondaryAccession] GDS851 Comment[ArrayExpressAccession] E-GEOD-1374 Comment[MAGETAB TimeStamp_Version] 2010-07-30 12:34:15 Last Changed Rev: 13058 Experimental Factor Name OrganismPart Experimental Factor Type organism_part Experimental Factor Term Source REF Person Last Name Budak Person First Name Murat Person Mid Initials T. Person Email mtbudak@mail.med.upenn.edu Person Phone Person Fax Person Address Rubinstein and Khurana Labs, University of Pennsylvania, Philadelphia, 19104, USA Person Affiliation University of Pennsylvania Person Roles submitter Person Roles Term Source REF The MGED Ontology 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 2007-10-31 PubMed ID 15467012 Publication DOI 15467012 Publication Author List Murat T Budak, Sasha Bogdanovich, Martin H J Wiesen, Olga Lozynska, Tejvir S Khurana, Neal A Rubinstein Publication Title Layer-specific differences of gene expression in extraocular muscles identified by laser-capture microscopy. Publication Status journal_article Publication Status Term Source REF The MGED Ontology Experiment Description Adult rat global and orbital layers of rectal extraocular muscles microdissected using Arcturus Pixcell II LCM are gene profiled here using affymetrix rat RAE230A gene chip. www.med.upenn.edu/cellbio/faculty/rubinstein/; www.uphs.upenn.edu/pmi/members/khurana/lab%20page.htm Protocol Name P-G1374-1 P-G1374-2 Protocol Type specified_biomaterial_action nucleic_acid_extraction Protocol Description Two adult Sprague-Dawley rats were killed using CO2 asphyxiation. Under sterile conditions, orbits were exenterated, and rectus EOM were quickly dissected out, placed in cryomolds, covered with OCT tissue embedding medium (Tissue-Tek; Sakura Finetek, Tokyo, Japan), and snap-frozen in 2-methylbutane chilled using liquid nitrogen. The EOM were then sectioned transversely into 7- to 10-μm sections using a Microm HM 500 cryostat (Zeiss, Oberkochen, Germany), mounted on SuperFrost Plus electrostatically charged slides (Fisher Scientific, Pittsburgh, PA), and immediately frozen on a dry ice. The sections were stored at –80°C until use. Every fifth slide was stained with hematoxylin and eosin to monitor overall tissue morphology. Sections taken from a region of EOM 1.3–2.3 mm anterior to the optic nerve ending were used for LCM. HistoGene LCM frozen section staining reagents were used for tissue dehydration and staining as described by the manufacturer (Arcturus Engineering, Mountain View, CA). Briefly, sections were fixed in 75% ethanol, hydrated in RNase-free water, and stained with HistoGene staining solution. Sections were washed in water and dehydrated sequentially in 75%, 95%, and 100% ethanol followed by 100% xylene. Sections were allowed to air dry and were stored in a desiccator at room temperature until utilized for LCM; care was taken to ensure that LCM was completed within 2 h of being placed in the desiccator.

The PixCell II LCM System (Arcturus Engineering) was used for LCM. A cap and thermoplastic film was placed above the frozen section. Areas of interest for microdissection were visualized using standard light microscopy; a single extremely brief pulse of laser beam ("zap") was used to melt the thin layer of thermoplastic film coating overlying a frozen section of tissue. To obtain sufficient amounts of material the procedure was repeated with the laser focused on different areas. Typically 3,000 zaps were performed per preparation (typically from 3–4 slides containing ~9–12 sections). The melted thermoplastic membrane and small region of tissue directly underneath (typically 10 μm2/zap) adhering to it were removed by means of lifting an overlying plastic cap. All animal experiments were approved by the University of Pennsylvania, Institutional Animal Care and Use Committee. Eight independent total RNA preparations were made from rectus muscles of two rats using the PicoPure RNA isolation kit (Arcturus Engineering). Briefly, total RNA was extracted from the captured cells by incubating LCM caps in extraction buffer for 30 min at 42°C. RNA was purified using preconditioned MiraCol purification columns. Eluted RNA was directly used for linear RNA amplification. Protocol Parameters Protocol Hardware Protocol Software Protocol Contact Protocol Term Source REF SDRF File E-GEOD-1374.sdrf.txt Term Source Name ncbitax nci_meta NCI_thesaurus The MGED Ontology ArrayExpress mo EFO The MGED Ontology Term Source File http://www.ncbi.nlm.nih.gov/Taxonomy/taxonomyhome.html http://ncimeta.nci.nih.gov/indexMetaphrase.html ncithesaurus.obo.alt 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