Hamilton Eye Institute (HEI) Retina Illumina V6.2 (Mar09) RankInv

Accession number: GN223

Summary:

HEI Retina Illumina V6.2 (Mar09) RankInv ** was uploaded by Arthur Centeno on March 25, 2009. This data set consists of 46 BXD strains, C57BL/6J, and both reciprocal F1s--49 strains total. No data for DBA/2J.

This data set has not been fully normalized. This is rank invariant data with 2z+8 stabilization, but without special correction for batch effects. The data includes the mean of four samples per strain. Values in expression range from 6.2 to 18.5 (12.3 units), a nominal range of 5000-fold.

The lowest level of expression is 6.25 for ILMN_2747167 from HEI Retina Illumina V6.2 (Mar09) RankInv **. Lowest single data about 5.7.

The highest level of expression is 18.50 for ILMN_2758581 (Gapdh). Highest single value is about 19.4.

Relevant Publications

1. Geisert EE, Lu L, Freeman-Anderson NE, Templeton JP, 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, (In Press)
2. 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
3. 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)
4. 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, 10(1):90.[Epub ahead of print] (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.

About the cases used to generate this set of data:

Almost all animals are young adults between 60 and 90 days of age (Table 1, minimum age is 48 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. Only one of these strains, BXD24 (know also known as BXD24b), has retinal degeneration (a spontaneous mutation). 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 the tissue used to generate this set of data:

Tissue preparation protocol. Animal were killed by rapid cervical dislocation. Retinas were removed immediately and placed in RNAlater at room temperature. Two retinas from one mouse were stored in a single tube.

Each array was hybridized with a pool of cRNA from 2 retinas (1 mouse). Natalie Freeman-Anderson extracted RNA at UTHSC.

Dissecting and preparing eyes for RNA extraction

Retinas for RNA extraction were placed in RNA STAT-60 (Tel-Test Inc.) and processed per manufacturer’s instructions (in brief form below). Total RNA was extracted with RNA STAT-60 (Tel-Test Inc.) according to the manufacturer's instructions. Briefly we:

• Homogenize tissue samples in the RNA STAT-60 (1 ml/50 to 100 mg tissue via syringe)
• Allow the homogenate to stand for 5-10 min at room temperature
• Add 0.2 ml of chloroform per 1 ml RNA STAT-60
• Mix the sample vigorously for 15 sec and let the sample incubate at room temperature for 5-10 min
• Centrifuge at 12,000 g for 30 min at 4°C
• Transfer the aqueous phase to a clean centrifuge tube
• Add 0.5 ml of isopropanol per 1 ml RNA STAT-60
• Vortex and incubate the sample at -20°C for 1 hr
• Centrifuge at 12,000 g for 30 min- 1 hr
• Remove the supernatant and wash the RNA pellet with 75% ethanol
• Remove ethanol, let air dry (5-10 min)
• Dissolve the pellet in 50 μl of nuclease free water.

Sample Processing Dr. Natalie Freeman-Anderson processed all samples in the HEI Vision Core Facility. The tissue was homogenized and extracted according to the RNA-Stat-60 protocol as described by the manufacturer (Tel-Test, Friendswood, TX) listed above. The quality and purity of RNA was assessed using an Agilent Bioanalyzer 2100 system. The RNA from each sample was processed with the Illumina TotalPrep RNA Amplification Kit (Ambion, Austin, TX) to produce labeled cRNA. The cRNA for each sample was then hybridized to an Illumina Sentrix® Mouse-6-V2 BeadChip (Illumina, San Diego, CA)

Once data was collected, we normalized the data using the formula 2 (z-score of log2 [intensity]) + 8 as previously described (Rogojina et al., 2003, Vazquez-Chona et al., 2004). This procedure sets the mean expression level across a single microarray to 8 units on an exponential scale similar to that produced by real-time qRT-PCR. For the microarray analysis, we compared the changes in the transcriptome of C57BL/6J mice to that of DBA/2J mice before and after optic nerve crush. The mice, at 60-90 days of age, could be considered adults with fully developed retinas. At this age range, DBA2/J mice had not yet developed symptoms associated with pigmentary dispersion glaucoma.

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.

Table 1: HEI Retina case IDs, including sample tube ID, strain, age, sex, and source of mice

About downloading this data set:

This data set is available as a bulk download in several formats. The data are available as either strain means or the individual arrays. Due to the involved normalization procedures required to correct for batch effects we strongly recommend not using the raw CEL files without special statistical procedures.

About the array platform:

Illumina MouseWG-6 v2.0 arrays: The Illumina Sentrix Mouse-6 BeadChip uses 50-nucleotide probes to interrogate approximately 46,000 sequences from the mouse transcriptome. For each array, the RNA was pooled from two retinas.

About data values and data processing:

Values of all 45,281 probe sets in this data set range from a low of 6.25 (Rho GTPase activating protein 11A, Arhgap11a, probe ID ILMN_2747167) to a high of 18.50 (glyceraldehyde-3-phosphate dehydrogenase, Gapdh, probe ID ILMN_2758581). This corresponds to 12.25 units or a 1 to 4900 dynamic range of expression (2^12.25). We normalized raw signal values using Beadstudio’s rank invariant normalization algorithm. BXD62 was the strain used as the control group

Normalization:

All normalization was performed by William E. Orr in the HEI Vision Core Facility

1. Computed the log base 2 of each raw signal value
2. Calculated the mean and standard Deviation of each Mouse WG-6 v2.0 array
3. Normalized each array using the formula, 2 (z-score of log2 [intensity]) The result is to produce arrays that have a mean of 8, a variance of 4, and a standard deviation of 2. The advantage is that a two-fold difference in expression level corresponds approximately to a 1 unit difference.
4. computed the mean of the values for the set of microarrays for each strain. Technical replicates were averaged before computing the mean for independent biological samples.

Data source acknowledgment:

Support for acquisition of microarray data sets was generously provided by Dr. Barrrett Haik, Chair of the Department of Ophthalmology, and Director of the Hamilton Eye Institute. Support for the continued development of GeneNetwork was provided by a NIDA/NIMH/NIAAA Human Brain Project grant, from funds from NEI grant to Dr. Eldon Geisert (R01EY017841), NEI Vision Core grant (EY14080), and an Unrestricted Grant from Research To Prevent Blindness.

Information about this text file:

Data set entered by Arthur Centeno, Sept 17, 2008. This text file originally generated by RWW and EEG. August 2009

References

Rogojina AT, Orr WE, Song BK, Geisert EE, Jr.: Comparing the use of Affymetrix to spotted oligonucleotide microarrays using two retinal pigment epithelium cell lines. Molecular vision 2003, 9:482-496.

Vazquez-Chona F, Song BK, Geisert EE, Jr.: Temporal changes in gene expression after injury in the rat retina. Investigative ophthalmology & visual science 2004, 45(8):2737-2746.

GEO Series Data: This section to be used for GEO submission of the whole series of arrays

GSE Series No GEO series number

Status Public on August, 2009

Organism(s) Mus musculus

Experiment type Expression profiling by array

Overall design We used pooled RNA samples of retinas, usually two independent pools--two male, two female pool--for most lines of mice.

Contributor(s) Eldon E. Geisert, Lu Lu, Natalie E. Freeman-Anderson, Robert W. Williams

Submission date Not yet submitted to GEO.
Contact name Eldon E. Geisert
E-mails
Phone 901-448-7740
FAX 901-448-5028
URL GeneNetwork BXD HEI RETINA
Organization name University of Tennessee Health Science Center
Department(s) Department of Ophthalmology
Laboratory(s) Geisert, Lu, Wiliams Labs
Street address 930 Madison Avenue
City Memphis
State/province TN
ZIP/Postal code 38163
Country USA

Platforms (1) GPLXXXX Illumina Mouse Whole Genome 6 version 2.0