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Summary
The DoD (Department of Defense) CDMRP (Congressionally Directed Medical Research Programs) Normal Retina Database uses the Affymetrix MouseGene 2.0 ST Array (May 15 2015) The RMA analysis and scaling was conducted by Arthur Centeno. This data set consists of 55 BXD strains, C57BL/6J, DBA/2J, a F1 cross between C57BL/6J and DBA/2J. A total of 58 strains were quantified. There is a total of 222 microarrays.
This is RMA expression data that has been normalized using what we call a 2z+8 scale, but without special correction for batch effects. The data for each strain was computed as the mean of four samples per strain. Expression values on a log2 scale range from 3.81 to 14.25 (10.26 units), a nominal range of approximately 1,000-fold. After taking the log2 of the original non-logged expression estimates, we convert data within an array to a z score. We then multiply the z score by 2. Finally, we add 8 units to ensure that no values are negative. The result is a scale with a mean expression of the probes on the array of 8 units and a standard deviation of 2 units. A two-fold difference in expression is equivalent roughly to 1 unit on this scale. The lowest level of expression is 3.81 (Olfr1186) from DoD CDMRP (Normal Retina Database uses the Affymetrix MouseGene 2.0 ST Array (May 15 2015) The highest level of expression is Rhodopsin for 17462036 (Rho). Highest single value is about 14.25.
Experiment design
ll of the procedures used involving mice were approved by IACUC at the Emory University and adhered to the ARVO Statement for the Use of Animals in Research. The Department of Defense (DoD) Congressionally Directed Medical Research Programs (CDMRP) Normal Retina Database uses the Affymetrix MouseGene 2.0 ST Array (May 15, 2015). Robust multiarray average (RMA) analysis and scaling were conducted by Arthur Centeno. This data set consists of 52 BXD strains, C57BL/6J, DBA/2J, and an F1 cross between C57BL/6J and DBA/2J. A total of 55 strains were quantified. There is a total of 222 microarrays. All data from each microarray used in this data set is publicly available on GeneNetwork.
These are RMA expression data that have been normalized using what we call a 2z+8 scale, but without corrections for batch effects. The data for each strain were computed as the mean of four samples per strain. The expression values on the log2 scale ranged from 3.81 to 14.25 (10.26 units), a nominal range of approximately 1,000-fold. After taking the log2 of the original non-logged expression estimates, we converted the data within an array to a z-score. We then multiplied the z-score by 2. Finally, we added 8 units to ensure that no values were negative. The result was a scale with the mean expression of the probes on the array of 8 units and a standard deviation of 2 units. A twofold difference in expression is equivalent to roughly 1 unit on this scale. The lowest level of expression was 3.81 (Olfr1186) from the DoD CDMRP (the Normal Retina Database uses the Affymetrix MouseGene 2.0 ST Array, May 15, 2015). The highest level of expression was rhodopsin for 17462036 (Rho). The highest single value was 14.25.
About cases
Almost all animals are young adults between 60 and 100 days of age (Table 1, minimum age is 60 and maximum age is 118 days). We measured expression in conventional inbred strains, BXD recombinant inbred (RI) strains, and reciprocal F1s between C57BL/6J and DBA/2J.
BXD strains: The first 32 of these strains are from the Taylor series of BXD strains generated at the Jackson Laboratory by Benjamin A. Taylor. BXD1 through BXD32 were started in the late 1970s, whereas BXD33 through 42 were started in the 1990s. BXD43 and higher were bred by Lu Lu, Jeremy Peirce, Lee M. Silver, and Robert W. Williams starting in 1997 using B6D2 generation 10 advanced intercross progeny. This modified breeding protocol doubles the number of recombinations per BXD strain and improves mapping resolution (Peirce et al. 2004). All of the Taylor series of BXD strains and many of the new BXD strains are available from the Jackson Laboratory. Several strains were specifically excluded from the dataset. For the BXD43 and higher, the DBA/2J parent carried both the Tyrp-1 mutation and the Gpnmb mutation and these two mutations produce pigment dispersion glaucoma. All of the mice carrying these two mutations were not included in the dataset: BXD53, BXD55, BXD62, BXD66, BXD68, BXD74, BXD77, BXD81, BXD88, BXD89, BXD95 and BXD98. In addition BXD 24 was omitted, since it developed a spontaneous mutation, rd16 (Cep290) which resulted in retinal degeneration and was renamed BXD24b/TyJ (ref). Several additional strains were excluded due to abnormally high Gfap levels observed in our Full HEI Retina (April 2010) dataset, these include: BXD32, BXD49, BXD70, BXD83 and BXD89.
About tissue
Tissue preparation protocol. Mice were killed by rapid cervical dislocation. Retinas were removed immediately and placed in 1 ml of 160 U/ml Ribolock for 1 min at room temperature. The retinas were removed from the eye and placed in Hank’s Balanced Salt solution with RiboLock in 50µl Ribolock (Thermo Scientific RiboLock RNase #EO0381 40U/µl 2500U) and stored in -80°C. The RNA was isolated using a QiaCube and the in column DNAse procedure. All RNA samples were checked for quality before running microarrays. The samples were analyzed using the Agilent 2100 Bioanalyzer. The RNA integrity values for ranged from 7.0 to 10. Our goal was to obtain data for independent biological sample pools from both sexes for most lines of mice. The four batches of arrays included in this final data set, collectively represent a reasonably well-balanced sample of males and females, in general without within-strain-by-sex replication.
About platform
Affymetrix Mouse Gene 2.0 ST Array: These expression arrays have been designed with a median of 22 unique probes per transcript. Each unique probe is 25 bases in length, which means that the array measures a median of 550 bases per transcript. The arrays provide comprehensive transcriptome coverage with over 30,000 coding and non-coding transcripts. In addition there is coverage for over 600 microRNAs. For some arrays the RNA was pooled from two retinas and for other arrays were run on a single retina. Dr. XiangDi Wang (UTHSC) and Becky King (Emory) were involved in the retinal extractions and isolation of RNA. The Affymetrix arrays were run by two different research cores: the Molecular Resource Center at UTHSC (Dr. William Taylor Director) and the Integrated Genomics Core at Emory University by Robert B Isett (Dr. Michael E. Zwick, Director). In a separate set of experiments we tested a set of arrays from C57BL/6J retinas run at each facility to determine if there were batch effects or other confounding differences in the results. We could not detect any significant difference in the arrays run at UTHSC or at Emory University. Thus, we have included both sets of data into the analysis.
Contributors
Rebecca King, Lu Lu, Robert W. Williams, Eldon E. Geisert
Citation
Transcriptome networks in the mouse retina: An exon level BXD RI database
Rebecca King,1 Lu Lu,2 Robert W. Williams,2 Eldon E. Geisert1
1Department of Ophthalmology and Emory Eye Center, Emory University, Atlanta, GA; 2Department of Anatomy and Neurobiology and Center for Integrative and Translational Genomics, University of Tennessee Health Science Center, Memphis, TN
Molecular Vision 2015; 21:1235-1251 http://www.molvis.org/molvis/v21/1235
Received 07 July 2015 | Accepted 22 October 2015 | Published 26 October 2015
Other Related Publications
1 Geisert EE, Lu L, Freeman-Anderson NE, Templeton JP, Nassr M, Wang X, Gu W, Jiao Y, Williams RW.:Gene expression in the mouse eye: an online resource for genetics using 103 strains of mice. Molecular Vision 2009 Aug 31;15:1730-63, (Link)
2. Freeman NE, Templeton JP, Orr WE, Lu L, Williams RW, Geisert EE (2011) Genetic networks in the mouse retina: Growth Associated Protein 43 and Phosphate Tensin Homology network. Molecular Vision 17:1355-1372.
3 Geisert EE, Jr., Williams RW: The Mouse Eye Transcriptome: Cellular Signatures, Molecular Networks, and Candidate Genes for Human Disease. In Eye, Retina, and Visual System of the Mouse. Edited by Chalupa LM, Williams RW. Cambridge: The MIT Press; 2008:659-674
4 Peirce JL, Lu L, Gu J, Silver LM, Williams RW: A new set of BXD recombinant inbred lines from advanced intercross populations in mice. BMC Genet 2004, 5:7. (Link)
5 Templeton JP, Nassr M, Vazquez-Chona F, Freeman-Anderson NE, Orr WE, Williams RW, Geisert EE: Differential response of C57BL/6J mouse and DBA/2J mouse to optic nerve crush. BMC Neurosci. 2009, July 30;10:90.(Link)
Other Data Sets Users of these mouse retina data may also find the following complementary resources useful:
1 NEIBank collection of ESTs and SAGE data.
2 RetNet: the Retinal Information Network--tables of genes and loci causing inherited retinal diseases
3 Mouse Retina SAGE Library from the Cepko laboratory. This site provides extensive developmental data from as early as embryonic day E12.5.
4 Digital reference of ophthalmology from Columbia provides high quality photographs of human ocular diseases, case studies, and short explanations. This reference does not have a molecular focus.
5 Mouse Retinal Developmental Gene Expression data sets from the Friedlander laboratory. This site provides extensive developmental data using the Affymetrix U74 v 2 array (predecessor of the M430).
6 Data sets on differential gene expression in anatomical compartments of the human eye from Pat Brown's lab. View expression signatures for different ocular tissues using the geneXplorer 2.0.
Acknowledgment
This work was supported by DoD CDMRP Grant W81XWH1210255 from the USA Army Medical Research & Materiel Command and the Telemedicine and Advanced Technology (EEG), NIH Grant R01EY017841 (EEG), Vision Core Grant P30EY006360 (P. Michael Iuvone), and Unrestricted Funds from Research to Prevent Blindness (Emory University). We thank XiangDi Wang and Arthur Centeno for their technical assistance in this project. This study was supported in part by the Emory Integrated Genomics Core (EIGC), which is subsidized by the Emory University School of Medicine and is one of the Emory Integrated Core Facilities.
Notes
This study includes Gene level and Exon level analysis.