HEI ONC Retina Illumina V6.2 (Sept11) RankInv (accession number: GN???)

Summary:

HEI ONC Retina Illumina V6.2 (Sept11) RankInv was normalized and scaled by William E. Orr and uploaded by Arthur Centeno and Xiaodong Zhou in September 2011. This data set consists of 57 BXD strains, C57BL/6J, DBA/2J, both reciprocal F1s, and BALB/cByJ. A total of 62 strains were quantified. The data are now open and available for analysis.

Please cite: Templeton JP, Wang XD, Freeman NE, Nickerson JM, Williams RW, Jablonski, MM, Rex, T, Geisert EE. Innate Immune Network in the Retina Activated by Optic Nerve Crush. (In process) (Link)

This is rank invariant 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 6.29 to 18.42 (12.13 units), a nominal range of approximately 4500-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 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 6.29 for ILMN_1225143 (Ust4r). Lowest single data about 5.93.

The highest level of expression is 18.42 for ILMN_2516699 (Ubb). Highest single value is about 19.78.

Other Related Publications

1. Freeman NE, Templeton JP, Orr WE, Lu L, Williams RW, Geisert EE: Genetic networks in the mouse retina: Growth Associated Protein 43 and Phosphate Tensin Homology network. Molecular Vision (2011) 17:1355-1372. (Link)
2. Jablonski MM, Freeman NE, Orr WE, Templeton JP, Lu L, Williams RW, Geisert EE: Genetic pathways regulating glutamate levels in retinal Muller cells. Neurochem Res. 2011 Apr;36(4):594-603. Epub 2010 Sep 30. (Link)
3. 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)
4. 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
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)
6. 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)

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 animals used to generate this set of data:

All animals are young adults between 60 and 90 days of age. 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 the tissue used to generate this set of data:

The Optic Nerve Crush (ONC) Method

Animal Use: All procedures were in compliance with institutional guidelines and with the ARVO statement for the Use of Animals in Ophthalmic and Vision Research. The Institutional Animal Care and Use Committee (IACUC) at the University of Tennessee Health Science Center approved all protocols involving the use of mice.

Anesthesia: The mice were anesthetized with a mixture of 13 mg/kg of Rompum and 87 mg/kg of Ketalar.

ONC Procedure: Under the binocular operating scope a small incision was made with the spring scissors (Roboz, cat. #RS-5619, Gaithersburg, MD) in the conjunctiva beginning inferior to the globe and around the eye temporally. With the micro-forceps (Dumont #5/45 Forceps, Roboz, cat. #RS-5005, Gaithersburg, MD), we grasped the edge of the conjunctiva and rotated the globe nasally, exposing the posterior aspect of the globe which allowed visualization the optic nerve. The exposed optic nerve was grasped approximately 1-3mm from the globe with Dumont #N7 cross action forceps (Roboz, cat. #RS-5027, Gaithersburg, MD) for 10 seconds, allowing the only pressure to be from the self-clamping action. After the 10 seconds the optic nerve is released and the forceps are removed allowing the eye to rotate back into place. The mice were allowed to recover on a warming pad.

Tissue preparation protocol. Two days after the ONC the 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.

Dr. Justin P. Templeton categorized the animals, as well as the ONC and retina removal.

Each array was hybridized with a pool of cRNA from 2 retinas (1 mouse). Dr. Clint Abner 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 1 hr 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 or overnight
• Centrifuge at 12,000 g for 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. Justin P. Templeton extracted the retinas from the mice and Drs. Clint Abner and Natalie Freeman 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)

Quality control analysis of the raw image data was performed using the Illumina BeadStudio software. MIAME standards were used for all microarray data. Rank invariant normalization with BeadStudio software was used to calculate the data. Once this data was collected, the data was globally normalized across all samples using the formula 2 (z-score of log2 [intensity]) + 8.

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 6.29, (integral membrane transport protein UST4r, Ust4r probe ID ILMN_1225143), to a high of 18.416 (Ubiquitin B, Ubb, probe ID ILMN_2516699). This corresponds to 12.13 units or a 1 to 4482.22 dynamic range of expression (2^12.13). 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.

Funding Support for the HEI Retina Dataset:

The HEI Retinal Database is supported by National Eye Institute Grants:

R01EY017841 (Dr. Eldon E. Geisert, PI)

P030EY13080 (NEI Vision Core Grant), and

A Unrestricted Grand from Research to Prevent Blindness (Dr. Barrett Haik, PI)

Information about this text file:

Dataset was uploaded to GeneNetwork by Arthur Centeno and Xiaodong Zhou, September 2011. This text file was generated by Justin P. Templeton January 2012.

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.(Link)

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.(Link)

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

GSE Series No GEO series number

Status Private on Sept, 2011

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, Justin P. Templeton, Robert W. Williams, Clint Abner, Natalie Freeman

Submission date Not yet submitted to GEO.
Contact name Eldon E. Geisert
E-mails EGeisert@uthsc.edu
Phone 901-448-7740
FAX 901-448-5028
URL GeneNetwork BXD HEI ONC 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