Comment[GEAAccession] E-GEAD-328 MAGE-TAB Version 1.1 Investigation Title Expression Profiling of developing eyes and brains in mouse embryos using NIG-NIA 15K mouse cDNA arrays Experiment Description The mechanisms of eye formation in the embryo vary according to developmental stage and adjacent tissues, especially the developing brain. These morphogenetic waves involve temporally interactions of transcription factors and spatially inductive signaling cascades. To throw light on the global genetic networks in regulating embryonic eye formation we used microarray hybridizations to profile the molecular changes occurring in the developing mouse eye between the stage of optic vesicle evagination at gestational day 9.5 (E9.5) and completion of basic eye formation at postnatal day 0 (P0). To display the differential molecular pattern of eye development in comparison to that of the developing brain. Experimental Design development or differentiation design Experimental Factor Name tissue Experimental Factor Type tissue Person Last Name Ronald Person First Name Wang Person Affiliation Research Institute, Osaka Medical Center for Cancer and Cardiovascular Diseases Person Roles submitter Public Release Date 2019-09-26 Protocol Name P-GEAD-279 P-GEAD-280 P-GEAD-281 P-GEAD-282 P-GEAD-283 P-GEAD-284 P-GEAD-285 Protocol Type sample collection protocol nucleic acid extraction protocol nucleic acid labeling protocol nucleic acid hybridization to array protocol array scanning and feature extraction protocol normalization data transformation protocol growth protocol Protocol Description none provided RNA in the whole embryo was preserved with RNAlater (Qiagen, West Sussex, United Kingdom) and then stored at -80 degree C. Total RNA was isolated using RNeasy Mini kits (Qiagen), and DNA was digested by DNA-free (Ambion, Austin, Texas). The presence of contaminating genomic DNA was excluded by PCR using S15 positive control primers. (Ambion) RNA quality was checked with gel electrophoresis and quantity was determined at OD260 in 50 mM sodium hydroxide. We prepared antisense RNA (aRNA) from 5 microg total RNA and 1 mol oligo-(dT)24-T7 (5'-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-3') primer. T7 Megascript Kit (Ambion) was employed for in vitro transcription, and RNeasy Mini kit (Qiagen) was used for aRNA purification and concentration. See also the array_protocol.pdf. Competitive hybridization was performed from aRNA of targets and aRNA of references. We labeled 2 microg aRNA together with 1 ng Arabidopsis CAB and RCP1 in vitro transcribed mRNAs (Stratagene, La Jolla, California) in a reverse transcription reaction with 3 microg of random hexamer primer (TaKaRa, Shiga, Japan). Both target and control aRNA in triplicate experiments were respectively labeled by 2 microl 1mM Cy3 or Cy5 (Amersham, Buckinghamshire, United Kingdom) dCTP; and 2 l 1mM Cy3 or Cy5 dUTP. The mixture was incubated at 42degree C for 2 h with 2 microl SSII (Invitrogen, Carlsbad, California) in 6 microl 5 first-strand buffer, 3 microl 0.1 M DTT, 1 microl RNasin, 3 microl 2 mM d(CT)TP and 5 mM d(AG)TP mix. To degrade the aRNA template, 10 microl 1M NaOH, 5 microl 0.5 M EDTA pH8.0 and 20 microl H2O were added; then, 25 microl 1 M Tri-HCl pH 8.0 was used to adjust pH in the mixture and the labelled targets were purified with Microcon 30 columns. (Millipore, Billerica, Massachusetts) before hybridisation. See also the array_protocol.pdf. none provided scanned on a ScanArray 4000 laser scanner (GSi Lumonica, Wilmington, Massachusetts). The resulting images were analyzed via QuantArray (GSi Lumonica) with a histogram algorithm and mean intensity calibration. Arabidopsis photosystem I chlorophyll a/b-binding protein (CAB) and root cap 1 (RCP1) (Stratagene) PCR products were used as external positive controls for intra- and inter-array calibrations; mouse transferrin receptor (TfR), glyceraldhyde-3-phosphate dehydrogenase (GAPDH) and beta-actin cDNAs were used as internal control for aRNA sequence specificity. Blank spots, poly(dA)45 oligonucleotide, Luciferase reporter pGL2 and a novel gene from Algae EST project (CIB, NIG, Japan) were used as negative controls. The array set (NIG-NIA 15K Mouse Array) consisted of 15,264 unique mouse probes and 976 control spots, with an estimated 0.05 microl of PCR products (1.0 microg/microl) picked up by 8 solid non-split pin system with phi 0.1 mm for each spot. Before hybridization, printed glass slides were treated with sodium borohydrate solution to ensure amnio-linkage of cDNA to the slides. See also the array_protocol.pdf. normalized to the reference standard by subtracting (in log space) the median observed value if it were other than zero. Fully-annotated, sequence verified and PCR validated cDNAs from the NIA 15K Mouse cDNA Clone Set (http://lgsun.grc.nia.nih.gov/cDNA/15k.html) were spotted onto poly-L-lysine-coated glass slides (Matsunami, Osaka, Japan), using a SPBIO 2000 spotter (Hitachi, Tokyo, Japan) in our spotting protocol. Plasmids were prepared with MontageTM Plasmid Miniprep96 Kit (Millipore) incorporated with Genesis 2100 Robotic Sample Processors (TECAN, Mannedorf, Switzerland). PCR-amplification of cDNAs was performed with 5' amnio-linked M13 forward primer. See also the array_protocol.pdf. Usual and normal house keeping SDRF File E-GEAD-328.sdrf.txt Comment[Number of channel] dual-channel Comment[Array Design REF] A-GEOD-10771 Comment[AEExperimentType] transcription profiling by array Comment[SecondaryAccession] CBX6 Comment[BioProject] PRJDB8815 Comment[CIBEX Accept Date] 2005-04-12 Comment[CIBEX Public Release Date] 2005-04-26 Comment[Last Update Date] 2019-09-26