Hippocampus Illumina NON (Oct08) RankInv beta**

Accession number: GN219

This is control expression data (No restraint and No injections, NON) for the four companion experimental data sets: No restraint and saline injection (NOS), No restraint and ethanol injection (NOE), Restraint stress and saline injection (RSS), and Restrain stress and ethanol injection (RSE).

This data set is a subset of the much larger Hippocampus Illumina (May07) RankInv data set. The subset was selected by Lu Lu to match the set of strains in the NOS, NOE, RSS, and RSE data sets. The NON subset uses the original Mouse 6.0 array and some probe sequences differ from the Mouse 6.1 array used for the experimental treatments.

WRONG ARRAY platform ID: This data set uses the ORGINAL Illumina Mouse 6.0 array.

    Summary:

May 07 ILLUMINA Mouse-6 DATA SET Rank Invariant Data Set: The LXS Hippocampus Illumina Rank Invariant data set provides estimates of mRNA expression in the hippocampus of 75 LXS recombinant inbred strains, the two parental strains, ILS/Ibg and ISS/Ibg (Inbred Long Sleep and Inbred Short Sleep strains from the Institute of Behavioral Genetics), and the two reciprocal F1 strains (ILSXISSF1, ISSXILSF1). All samples are from normal adult control animals raised in a standard laboratory environment. Subsequent data sets will provide estimates of mRNA expression following restraint stress, ethanol treatment, and stress followed by ethanol using many of the same strains (Lu Lu and colleagues).

A total of 240 pooled hippocampal samples were processed using 40 Illumina Sentrix Mouse-6 v 1.0 oligomer microarray BeadArray slides. Twenty-seven Mouse-6 slides and a total of 160 samples passed stringent quality control and error checking. We should note that this is our first experience using the Illumina platform and the initial set of 13 slides were are not included. This particular data set was processed using the Illumina "Rank Invariant" protocol. Values were log2 transformed and the current data range from 6.141 average (very low or no expression) to 19.987 (extremely high).

As a measure of data quality we often count the number of probes that are associated with LOD scores of greater than 10 (LRS > 46). In this Hippocampus Illumina (May 07) RankInv data set, 1183 probes have LRS values >46.

In comparison, here are the yields of QTLs with LOD>10 for other closely related data sets:

1. NO DATA for Hippocampus Illumina (Aug07) RSN
2. NO DATA for Hippocampus Illumina (Aug07) RSN_NB
3. 1050 for Hippocampus Illumina (Aug07) LOESS
4. 1162 for Hippocampus Illumina (Aug07) LOESS_NB
5. 1129 for Hippocampus Illumina (Aug07) QUANT
6. 1176 for Hippocampus Illumina (Aug07) QUANT_NB
7. 1183 for Hippocampus Illumina (May 07) RankInv (THIS DATA SET)
8. 1167 for Hippocampus Illumina (Oct06) Rank
9. 1170 for Hippocampus Illumina (Oct06) RankInv

The LRS achieved in the different version of the LXS Hippocampus data for probe ILM103520706 (Disabled 1; Dab1) are as follows:

1. 374.8 for Hippocampus Illumina (Aug07) RSN
2. 363.0 for Hippocampus Illumina (Aug07) RSN_NB
3. 338.4 for Hippocampus Illumina (Aug07) LOESS
4. 339.8 for Hippocampus Illumina (Aug07) LOESS_NB
5. 370.2 for Hippocampus Illumina (Aug07) QUANT
6. 363.5 for Hippocampus Illumina (Aug07) QUANT_NB
7. 360.3 for Hippocampus Illumina (May 07) RankInv
8. 358.1 for Hippocampus Illumina (Oct06) Rank
9. 358.8 for Hippocampus Illumina (Oct06) RankInv

Legend: UPDATE FIGURE: Bar chart of the expression of Dab1 probe ILM103520706 in the LXS data set. This probe has a Mendelian segregation pattern and is associated with an LRS 360.3 in this May07 RankInv data set vs 358.8 for the previous Oct06 data set.

ABOUT THE HIPPOCAMPUS. The hippocampus is an important and intriguing part of the forebrain that is crucial in memory formation and retrieval. This region of the brain is particularly vulnerable to the effects of environmental stressors and is a key upstream modulator of the hypothalamic-pituitary-adrenal axis (the HPA). The hippocampus is also often affected in epilepsy, Alzheimer's disease, and schizophrenia. Unlike most other parts of the brain, the hippocampus contains a remarkable population of stems cells that continue to generate neurons and glial cells even in adult mammals (Kempermann, 2005). This genetic analysis of transcript expression in the hippocampus (dentate gyrus, CA1-CA3) is a joint effort of 14 investigators supported by numerous agencies described in the Acknowledgments section.

     About the strains used to generate this set of data:

The LXS genetic reference panel of recombinant inbred strains consists of just over 77 strains. All of these strains have been inbred for more than 23 generations (F23). All strains have been genotyped at 13,377 SNPs. Thanks to the efforts of Dr. Timothy Wiltshire at the Genome Institute of the Novartis Research Foundation, the two parental strains have been genotyped at 156,551 SNPs. These genotypes are incorporated in the GeneNetwork SNP Browser.

Strains are currently available from Drs. Beth Bennett and Tom Johnson at the Institute of Behavioral Genetics (IBG) in Boulder Colorado.

    About the animals and tissue used to generate this set of data:

All animals were raised at the IBG by Bennett and colleagues in an SPF facility. No cases were MHV positive. Mice were killed by cervical dislocation. Whole brain dissections were performed at the IBG by Bennett and colleagues and shipped in RNAlater to Lu Lu and colleagues at UTHSC. Most hippocampal dissections (all were bilateral) were performed by Zhiping Jia. Cerebella, olfactory bulbs, and brain stems were also dissected and stored at -80 deg C using further use. Hippocampal samples are very close to complete (see Lu et al., 2001 but probably include variable amounts of fimbria and choroid plexus (see expression of transthyretin, Ttr, as a marker of choroid plexus).

A pool of dissected tissue from four hippocampi taken from two naive adults of the same strain, sex, and age was collected in one session and used to generate RNA samples. The great majority (75%) of animals were sacrificed between 9:30 AM and 11:30 AM. All animals were sacrificed between 9 AM and 5 PM during the light phase. All RNA samples were extracted at UTHSC by Zhiping Jia.

All animals used in this study were between 53 and 90 days of age (average of 72 days; see Table 1 below).

Sample Processing: Samples were processed by Lu Lu and colleagues in the Illumina Core at UTHSC between July 25 and Dec 20, 2006. All processing steps were performed by Feng Jiao. In brief, RNA purity was evaluated using the 260/280 nm absorbance ratio, and values had to be greater than 1.8 to pass our quality control (QC). The majority of samples had values between 1.9 and 2.1. RNA integrity was assessed using the Agilent Bioanalyzer 2100. The standard Eberwine T7 polymerase method was used to catalyze the synthesis of cDNA template from polyA-tailed RNA using the Ambion/Illumina (http://www.ambion.com/catalog/CatNum.php?AMIL1791) TotalPrep RNA amplication kit (Cat#IL1791). The biotin labeled cRNA was then evaluated using both the 260/280 ratio (values of 2.0-2.3 are acceptable) using a NanoDrop ND-1000 (http://www.nanodrop.com/nd-1000-overview.html). Those samples that passed QC steps (1-3% failed and new RNA samples had to be acquired and processed) were immediately used on the original Mouse-6 v 1.0 slide. The slides were hybridized and washed following standard Illumina protocols.

Replication and Sample Balance: We obtained a male sample pool and female sample pool from each strain. While all strains were orginally represented by matched male and female samples, one strain LXS34 consists of two female samples. Given the expression of Xist, we suspect that strain LXS114 is represented by two male pools (see figure at bottom of page).

Experimental Design and Batch Structure: This data set consists arrays processed in 13 groups over a five month period (July 2006 to Dec 2006). Most groups consisted of 12 samples. All arrays in this data set were processed using a single protocol by a single operator, Feng Yiao. Processing was supervised directly by Dr. Lu Lu. All samples were scanned on a single Illumina Beadstation housed in the Hamilton Eye Institute between July 28 and Dec 21, 2006. Details on sample assignment to slides and batches is provide in the table below.

    Data Table 1:

This table lists all arrays by order of strain (index) and includes data on tube ID, strain, age, sex, F generation number, number of animals in each sample pool (pool size), slide ID, slide position (A through F), scan date, and scan batch.

    Downloading all data:

All data links (right-most column above) will be made active as soon as the global analysis of these data has been accepted for publication. Please see text on Data Sharing Policies, and Conditions and Limitations, and Contacts. Following publication, download a summary text file or Excel file of data. Please contact Dr. Lu Lu if you have any questions on the use of these open data.

    About the array platform:

Illumina Sentrix Mouse-6 BeadArray Platform: The Mouse6 array consists of 46,116 unique probe sequences, each 50 nucleotides in length, that have been arrayed on glass slides using a novel bead technology.

Dunning M, Smith M, Thorne N, Tavare S (2006) beadarray: An R package to analyse Illumina BeadArrays. R News (the Newsletter of the P Project) 6:17-23. (see pages 17-23 of http://CRAN.R-project.org/doc/Rnews/Rnews_2006-5.pdf).

ANNOTATION: In spring of 2007, Robert W. Williams and Hongqiang Li reannotated the Illumina Mouse-6 array content. This new annotation is now incorporated into GeneNetwork. For 46166 probes on the Mouse 6 array platform (including control probes) we have identified 35975 NCBI Entrez Gene IDs; 26481 matched human Gene IDs; 23899 matched rat Gene IDs; 26883 NCBI HomoloGene IDs; and 12791 OMIM IDs.

Position data for the 50-mer Illumina Mouse-6 array were initially downloaded from Sanger at http://www.sanger.ac.uk/Users/avc/Illumina/Mouse-6_V1.gff.gz but we then updated all positions by BLAT analysis from mm6 positions to mm8 positions (Hongqiang Li).

    About data processing:

This data set uses the standard Rank Invariant method developed by Illumina and described in their BeadStation Studio documentation.

Sex of the samples was validated using sex-specific probe set: Xist (probe ILM106520068, also known as scl00213742.1_141-S).

Legend: Checking that the sex of samples was labeled correctly in mouse array data sets using Xist expression measured by probe ILM106520068. In this bar chart the expression of Xist is very low in LXS114 and has a low error term. This is because both arrays are male samples rather than 1 male and 1 female sample. In contrast LXS34 has very high expression and no error bar because the sample is from a single female pool.

    Data source acknowledgment:

Data were generated with funds to Lu Lu, Beth Bennett, Mike Miles, Melloni Cook from INIA.

Lu Lu, M.D.
Grant Support: NIH U01AA13499, U24AA13513 (Lu Lu, PI)

    About this text file:

Data uploaded by Hongqiang Li, Oct 30, 2006. This text file originally generated by LL and RWW on November 29, 2006. Updated by LL, Dec 1, 2006. Updated March 25, April 25 by RWW. Updated with data on LOD scores, Oct 24, 2007 by RWW>