DoD Retina Blast vs Normal Retina Affy MoGene 2.0 ST (Apr16) RMA Exon Level

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DoD TATRC Retina Dataset Affymetrix MouseGene 2.0 ST Array (____ 2015) RMA analysis and scaling by Arthur Centeno. This data set consists of 75 BXD strains, C57BL/6J, DBA/2J, both reciprocal F1s, and BALB/cByJ. A total of 80 strains were quantified. The data are now open and available for analysis.

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 6.25 to 18.08 (11.83 units), a nominal range of approximately 3600-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 6.254 for ILMN_2747167 (Arhgap11a) from HEI Retina Illumina V6.2 (April 2010) RankInv **. Lowest single data about 5.842. The highest level of expression is 18.077 for ILMN_2516699 (Ubb). Highest single value is about 18.934.

Experiment design

Sample Processing: 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 could not detect any significant difference in the arrays run at UTHSC or at Emory University.

Replication, sex, and sample balance: 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 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.
  • In 2004, BXD24/TyJ developed a spontaneous mutation, rd16 which resulted in retinal degeneration and was renamed BXD24b/TyJ (BXD24 in this database). The strain, BXD24a, was cryo-recovered in 2004 from 1988 embryo stocks (F80) and does not exhibit retinal degeneration. In 2009, BXD24b was renamed BXD24/TyJ-Cep290rd16/J by JAX Labs to reflect the discovery of the genetic basis of the mutation. At the same time BXD24a was then referred to just as BXD24/TyJ by Jax Labs, but still called BXD24a in this dataset.
  • The other 36 BXD strains (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. All of the new BXD strains (BXD43 and higher) are also available directly from Lu Lu and colleagues at the University of Tennessee Health Science Center in Memphis, TN, USA. BXD24/TyJ is now known as BXD24b/TyJ and has nearly complete retinal degeneration. BXD24a/TyJ, a 1988 F80 inbred stock that has been rederived from cryogenic storage, does not have retinal degeneration (stock number 005243) and is an ideal coisogenic control, but is not included in the HEI data set.

About tissue

Tissue preparation protocol. Animal 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. Dissecting and preparing eyes for RNA extraction Retinas for RNA removed from the eye and placed in Hank’s Balanced Salt solution with RiboLock (Thermo Scientific RiboLock RNase #EO0381 40U/µl 2500U) and processed per manufacturer’s instructions (in brief form below).

1) Sample collection for RNA isolation.

2) Quickly remove the retinas with clean curved forceps after cervical dislocation of the mouse.

3) Put each retina in 1 ml of 160 U/ml Ribolock for 1 min in RT.

4) Move the retina to another tube with 50µl Ribolock, store in -80°C.

5) The RNA was isolated using a QiaCube and the in column DNAse procedure.

Quality Control: All RNA samples were checked for quality before running microarrays. The samples were analyzed using the Agilent 2100 Bioanalyzer. The RNA integrity values for each sample are presented in Table 1 below.

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 592 microRNAs. For some arrays the RNA was pooled from two retinas and for other arrays were run on a single retina.


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.

Specifics of this data set

Exon Level