# HBP Rosen Striatum M430V2 (Apr05) RMA ^{}

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## Summary

This April 2005 data freeze provides estimates of mRNA expression in the striatum (caudate nucleus of the forebrain) of 33 lines of mice including C57BL/6J, DBA/2J, and 31 BXD recombinant inbred strains. Data were generated using Affymetrix Mouse Genome 430 2.0 short oligomer microarrays at Beth Israel Deaconess Medical Center (BIDMC, Boston MA) by Glenn D. Rosen with the support of a Human Brain Project (HBP) grant. Approximately 250 brain samples (males and females) from 33 strains were used in this experiment. Samples were hybridized to a total of 59 arrays. This particular data set was processed using the Microarray Suite 5 protocol (MAS 5). To simplify comparison among different transforms, MAS5 values of each array have been adjusted to an average expression of 8 units and a standard deviation of 2 units.

## Experiment design

RNA was extracted by Rosen and colleagues and was then processed by the BIDMC Genomics Core. Labeled cRNA was generated using the Amersham Biosciences cRNA synthesis kit protocol.

Replication and Sample Balance:Our goal is to obtain data for independent biological sample pools from at least one sample from each sex for all BXD strains. We have not yet achieved this goal. Twenty-three of 33 strains are represented by male and female samples. The remaining 8 strains are still represented by single sex samples: BXD11 (F), BXD13 (F), BXD19 (F), BXD20 (F), BXD22 (M), BXD23 (M), BXD24 (M), BXD32 (M), C57BL/6J (M), and DBA/2J (M).

Batch Structure:This data set consists of arrays processed in three batches with several reruns for the first batch. All arrays were processed using a single protocol. All data have been corrected for batch effects as described below.

## About cases

We have used a set of BXD recombinant inbred strains generated by crossing C57BL/6J (B6 or B) with DBA/2J (D2 or D). The BXDs are particularly useful for systems genetics because both parental strains have been sequenced (8x coverage of B6 and 1.5x coverage for D). Physical maps in WebQTL incorporate approximately 1.75 million B vs D SNPs from Celera. BXD2 through BXD32 were bred by Benjamin A. Taylor starting in the late 1970s. BXD33 through 42 were bred by Taylor in the 1990s. These strains are available from The Jackson Laboratory.

## About tissue

Animals were obtained from The Jackson Laboratory and housed for several weeks at BIDMC until they reached ~2 months of age (range from 55 to 62 days). Mice were killed by cervical dislocation and brains were removed and placed in RNAlater for 20 to 25 minutes prior to dissection. Cerebella and olfactory bulbs were removed; brains were hemisected, and both striata were dissected using a medial approach by GD Rosen that typically yields 5 to 7 mg of tissue per side. The purity of this dissection has been validated by an analysis of acetylcholinestase activity. A pool of dissected tissue from 3 or 4 adults (approximately 25 to 30 mg of tissue) of the same strain, sex, and age was collected in one session and used to generate cRNA samples.

The table below lists the arrays by strain, sex, sample name, and batch ID. Each array was hybridized to a pool of mRNA from 3 to 4 mice. All mice were between 55 and 62 days.

Id Strain Sex Sample_name BatchId 1 C57BL/6J M Chip41_Batch02_B6_M_Str Batch02 2 C57BL/6J M Chip11_Batch03_B6_M_Str Batch03 3 BXD1 F Chip03_Batch03_BXD1_F_Str Batch03 4 BXD1 M Chip04_Batch03_BXD1_M_Str Batch03 5 BXD2 F Chip20_Rerun01_BXD2_F_Str Rerun01 6 BXD2 M Chip05_Batch01_BXD2_M_Str Batch01 7 BXD5 F Chip10_Batch03_BXD5_F_Str Batch03 8 BXD5 M Chip12_Batch03_BXD5_M_Str Batch03 9 BXD6 F Chip38_Batch02_BXD6_F_Str Batch02 10 BXD6 M Chip39_Batch02_BXD6_M_Str Batch02 11 BXD8 F Chip07_Batch03_BXD8_F_Str Batch03 12 BXD8 M Chip02_Batch03_BXD8_M_Str Batch03 13 BXD9 F Chip16_Batch01_BXD9_F_Str Batch01 14 BXD9 M Chip10_Batch01_BXD9_M_Str Batch01 15 BXD11 F Chip31_Batch02_BXD11_F_Str Batch02 16 BXD12 F Chip11_Batch01_BXD12_F_Str Batch01 17 BXD12 M Chip18_Batch03_BXD12_M_Str Batch03 18 BXD13 F Chip33_Batch02_BXD13_F_Str Batch02 19 BXD14 F Chip48_Batch02_BXD14_F_Str Batch02 20 BXD14 M Chip47_Rerun01_BXD14_M_Str Rerun01 21 BXD15 F Chip21_Batch01_BXD15_F_Str Batch01 22 BXD15 M Chip13_Batch01_BXD15_M_Str Batch01 23 BXD16 F Chip36_Batch02_BXD16_F_Str Batch02 24 BXD16 M Chip44_Rerun01_BXD16_M_Str Rerun01 25 BXD18 F Chip15_Batch03_BXD18_F_Str Batch03 26 BXD18 M Chip19_Batch03_BXD18_M_Str Batch03 27 BXD19 F Chip19_Batch01_BXD19_F_Str Batch01 28 BXD20 F Chip14_Batch03_BXD20_F_Str Batch03 29 BXD21 F Chip18_Batch01_BXD21_F_Str Batch01 30 BXD21 M Chip09_Batch01_BXD21_M_Str Batch01 31 BXD22 M Chip13_Batch03_BXD22_M_Str Batch03 32 BXD23 M Chip01_Batch03_BXD23_M_Str Batch03 33 BXD24 M Chip17_Batch03_BXD24_M_Str Batch03 34 BXD27 F Chip29_Batch02_BXD27_F_Str Batch02 35 BXD27 M Chip40_Batch02_BXD27_M_Str Batch02 36 BXD28 F Chip06_Batch01_BXD28_F_Str Batch01 37 BXD28 M Chip23_Batch01_BXD28_M_Str Batch01 38 BXD29 F Chip45_Batch02_BXD29_F_Str Batch02 39 BXD29 M Chip42_Batch02_BXD29_M_Str Batch02 40 BXD31 F Chip14_Batch01_BXD31_F_Str Batch01 41 BXD31 M Chip09_Batch03_BXD31_M_Str Batch03 42 BXD32 M Chip30_Batch02_BXD32_M_Str Batch02 43 BXD33 F Chip27_Rerun01_BXD33_F_Str Rerun01 44 BXD33 M Chip34_Batch02_BXD33_M_Str Batch02 45 BXD34 F Chip03_Batch01_BXD34_F_Str Batch01 46 BXD34 M Chip07_Batch01_BXD34_M_Str Batch01 47 BXD36 F Chip22_Batch03_BXD36_F_Str Batch03 48 BXD36 M Chip24_Batch03_BXD36_M_Str Batch03 49 BXD38 F Chip17_Batch01_BXD38_F_Str Batch01 50 BXD38 M Chip24_Batch01_BXD38_M_Str Batch01 51 BXD39 M Chip20_Batch03_BXD39_M_Str Batch03 52 BXD39 F Chip23_Batch03_BXD39_F_Str Batch03 53 BXD39 M Chip43_Rerun01_BXD39_M_Str Rerun01 54 BXD40 F Chip08_Rerun01_BXD40_F_Str Rerun01 55 BXD40 M Chip22_Batch01_BXD40_M_Str Batch01 56 BXD42 F Chip35_Batch02_BXD42_F_Str Batch02 57 BXD42 M Chip32_Batch02_BXD42_M_Str Batch02 58 DBA/2J M Chip02_Batch01_D2_M_Str Batch01 59 DBA/2J M Chip05_Batch03_D2_M_Str Batch03

## About platform

Affymetrix Mouse Genome 430 2.0 array:The 430v2 array consists of 992936 useful 25-nucleotide probes that estimate the expression of approximately 39,000 transcripts (many are near duplicates). The array sequences were selected late in 2002 using Unigene Build 107. The array nominally contains the same probe sequence as the 430A and B series. However, we have found that roughy 75000 probes differ from those on A and B arrays.

## About data processing

**Probe (cell) level data from the CEL file: **These CEL values produced by GCOS are 75% quantiles from a set of 91 pixel values per cell.

- Step 1: We added an offset of 1.0 unit to each cell signal to ensure that all values could be logged without generating negative values. We then computed the log base 2 of each cell.
- Step 2: We performed a quantile normalization of the log base 2 values for the total set of 105 arrays (processed as two batches) using the same initial steps used by the RMA transform.
- Step 3: We computed the Z scores for each cell value.
- Step 4: We multiplied all Z scores by 2.
- Step 5: We added 8 to the value of all Z scores. The consequence of this simple set of transformations is to produce a set of Z scores that have a mean of 8, a variance of 4, and a standard deviation of 2. The advantage of this modified Z score is that a two-fold difference in expression level corresponds approximately to a 1 unit difference.
- Step 6: We eliminated much of the systematic technical variance introduced by the batches at the probe level. To do this we calculated the ratio of each batch mean to the mean of both batches and used this as a single multiplicative probe-specific batch correction factor. The consequence of this simple correction is that the mean probe signal value for each batch is the same.
- Step 7: Finally, we computed the arithmetic mean of the values for the set of microarrays for each strain. Technical replicates were averaged before computing the mean for independent biological samples. Note, that we have not (yet) corrected for variance introduced by differences in sex or any interaction terms. We have not corrected for background beyond the background correction implemented by Affymetrix in generating the CEL file. We eventually hope to add statistical controls and adjustments for some of these variables.

**Probe set data: **The expression data were processed by Yanhua Qu (UTHSC). Probe set data were generated from the fully normalized CEL files (quantile and batch corrected) using the standard MAS 5 Tukey biweight procedure. A 1-unit difference represents roughly a two-fold difference in expression level. Expression levels below 5 are usually close to background noise levels. **Data quality control: **A total of 62 samples passed RNA quality control.

Probe level QC: Log2 probe data of all arrays were inspected in DataDesk before and after quantile normalization. Inspection involved examining scatterplots of pairs of arrays for signal homogeneity (i.e., high correlation and linearity of the bivariate plots) and looking at all pairs of correlation coefficients (62x61/2). Arrays with probe data that was not homogeneous when compared to any other arrays was flagged. If the correlation at the probe level was less than approximately 0.92 we deleted that array data set. Three arrays we lost during this process (BXD19_M_Str_Batch03, BXD23_F_Str_Batch03, and BXD24_F_Str_Batch03).

Probe set level QC: The final normalized strain averages were evaluated for outliers. This involved counting the number of times that the probe set value for a particular strain was beyond two standard deviations of the mean of all strains. (We used the PDNN transform as our reference probe set data for this QC step.) Two strains, each represented by single arrays, generated greater than 5,000 outlier counts (10% of the number of probe sets). These two arrays generated a great number of outliers across the entire range of expression and since we do not yet have replicate arrays for either of these two strains we opted to delete them from the final April 2005 striatum data sets.

## Acknowledgment

Data were generated with funds to Glenn Rosen from P20 MH62009 (see below for specifics). Samples and arrays were processed by the Genomics Core at Beth Israel Deaconess Medical Center by Towia Libermann and colleagues.

## Notes

This text file originally generated by GDR, RWW, and YHQ on Nov 2004. Updated by RWW Nov 17, 2004; GDR and RWW, Dec 23, 2004; RWW and GDR April 8, 2005.