INIA M430 brain RMA Database (April/05 Freeze) modify this page

Accession number: GN59

    Summary:

This April 2005 data freeze provides estimates of mRNA expression in adult forebrain and midbrain from 45 lines of mice including C57BL/6J, DBA/2J, their F1 hybrids, and 42 BXD recombinant inbred strains. Data were generated at UTHSC and the University of Memphis with support from grants from the NIAAA Integrative Neuroscience Initiative on Alcoholism (INIA). Samples were hybridized in small pools (n = 3) to a total of 105 Affymetrix M430A and B array pairs. This particular data set was processed using the RMA protocol. To simplify comparisons among transforms, RMA values of each array were adjusted to an average of 8 units and a standard deviation of 2 units.

    About the cases used to generate this set of data:

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. BXD43 through BXD99 were bred by Lu Lu, Jeremy Peirce, Lee M. Silver, and Robert W. Williams in the late 1990s and early 2000s using advanced intercross progeny (Peirce et al. 2004). Many of the 50 new BXD strains are available from Lu Lu and colleagues

All stock was obtained originally from The Jackson Laboratory between 1999 and 2003. Most BXD animals were born and housed at the University of Tennessee Health Science Center. Some cases were bred at the University of Memphis (Douglas Matthews) or the University of Alabama (John Mountz and Hui-Chen Hsu).

    About the tissue used to generate this set of data:

The INIA M430 brain Database (April05) consists of 105 Affymetrix 430A and 430B microarray pairs. Each pair was hybridized in sequence (A array first, B array second) with a pool of brain tissue (forebrain minus olfactory bulb, plus the entire midbrain) taken from three adult animals of closely matched age and the same sex. RNA was extracted at UTHSC by Lu Lu, Zhiping Jia, and Hongtao Zhai. All samples were subsequently processed in the INIA Bioanalytical Core at the W. Harry Feinstone Center of Excellence by Thomas R. Sutter, Shirlean Goodwin, and colleagues at the University of Memphis.

Replication and Sample Balance: Our goal is to obtain data for independent biological sample pools from at least one of sample from each sex for all BXD strains. We have not yet achieved this goal. Ten of 45 strains are still represented by single sex samples: BXD2 (F), BXD8 (F), BXD15 (F), BXD18 (F), BXD25 (F), BXD29 (F), BXD33 (M), BXD45 (F), BXD77 (M), and BXD90 (M). Eleven strains are represented by three independent samples with the following breakdown by sex: C57BL/6J (1F 2M), DBA/2J (2F 2M), B6D2F1 (2F 2M) + D2B6F1 (1F 1M), BXD6 (2F 1M), BXD13 (2F 1M), BXD14 (1F 2M), BXD28 (2F 1M), BXD34 (1F 2M), BXD36 (1F 2M), BXD38 (1F 2M), BXD42 (1F 2M).

Batch Structure: Before running the first batch of 30 pairs of array (dated Jan04), we ran four test samples (Nov03). The main batch of 30 includes the four test samples (four technical replicates). The Nov03 data was combined with the Jan04 data and was treated as a single batch that consists of one male and one female pool from C57BL/6J, DBA/2J, the B6D2F1 hybrid, 11 female BXD samples, and 11 male BXD samples. The second large batch was run February 2005 (Feb05) and consists of 71 pairs of arrays. Batch effects were corrected at the individual probe level as described below.

The table below summarizes information on strain, sex, age, sample name, batch result date, and source of mice.

IdStrain Sex Age Sample_name Result dateSource
1C57BL/6JF65R0903F1Nov03UTM RW
2C57BL/6JF65R0903F1Jan04UTM RW
3C57BL/6JM66R0906F1Nov03UTM RW
4C57BL/6JM66R0906F1Jan04UTM RW
5C57BL/6JM66R0906F1Feb05UTM RW
6C57BL/6JM76R0997F1Feb05UTM RW
7D2B6F1F57R1066F1Feb05UTM RW
8D2B6F1M59R1381F1Feb05UTM RW
9DBA/2JF60R0917F1Nov03UTM RW
10DBA/2JF60R0917F1Feb05UTM RW
11DBA/2JF60R0917F2Jan04UTM RW
12DBA/2JF64R1123F1Feb05UTM RW
13DBA/2JM60R0918F1Nov03UTM RW
14DBA/2JM60R0918F1Jan04UTM RW
15DBA/2JM73R1009F1Feb05UTM RW
16B6D2F1F127R0919F1Jan04UTM JB
17B6D2F1F127R0919F2Jan04UTM JB
18B6D2F1F64R1053F1Feb05UTM RW
19B6D2F1F64R1053F1Feb05UTM RW
20B6D2F1M127R0920F1Jan04UTM JB
21B6D2F1M127R0920F2Jan04UTM JB
22B6D2F1M66R1057F1Feb05UTM RW
23BXD1M181R0956F1Feb05UTM JB
24BXD1F95R0895F1Jan04UMemphis
25BXD2F142R0907F1Feb05UAB
26BXD5F56R0744F1Feb05UMemphis
27BXD5M71R0728F1Jan04UMemphis
28BXD6F57R1711F1Feb05JAX
29BXD6F92R0901F1Feb05UMemphis
30BXD6M92R0902F1Jan04UMemphis
31BXD8F72R0167F1Jan04UAB
32BXD9F86R0908F1Feb05UMemphis
33BXD9M86R0909F1Jan04UMemphis
34BXD11F97R0745F1Feb05UAB
35BXD11M92R0666F1Feb05UMemphis
36BXD12F64R0896F1Feb05UMemphis
37BXD12M64R0897F1Jan04UMemphis
38BXD13F86R0730F1Feb05UMemphis
39BXD13F86R0748F1Jan04UMemphis
40BXD13M76R0929F1Feb05UMemphis
41BXD14M91R0912F1Jan04UMemphis
42BXD14M68R1051F1Feb05UTM RW
43BXD15F80R0928F1Feb05UMemphis
44BXD18F108R0771F1Jan04UAB
45BXD19M157R1229F1Feb05UTM JB
46BXD19F56R0236F1Jan04UAB
47BXD21F67R0740F1Jan04UAB
48BXD21F67R0740F1Feb05UAB
49BXD23F66R1035F1Feb05UTM RW
50BXD23M66R1037F1Feb05UTM RW
51BXD23F88R0815F1Jan04UAB
52BXD23F88R0815F1Feb05UAB
53BXD24F71R0914F1Feb05UMemphis
54BXD24M71R0913F1Jan04UMemphis
55BXD25F74R0373F1Jan04UTM RW
56BXD28F79R0910F1Jan04UMemphis
57BXD28M79R0911F1Feb05UMemphis
58BXD28F113R0892F1Feb05UTM RW
59BXD29F76R0693F1Jan04UMemphis
60BXD31F61R1199F1Feb05UTM RW
61BXD31M61R1141F1Feb05UTM RW
62BXD32F93R0898F1Jan04UAB
63BXD32F76R1214F1Feb05UMemphis
64BXD32M65R1478F1Feb05UMemphis
65BXD33M77R0915F1Jan04UMemphis
66BXD34F92R0900F1Feb05UMemphis
67BXD34M56R0617F1Feb05UMemphis
68BXD34M72R0916F1Jan04UMemphis
69BXD36F61R1145F1Feb05UTM RW
70BXD36M77R0926F1Jan04UMemphis
71BXD36M61R1211F1Feb05UMemphis
72BXD38M83R1208F1Feb05UMemphis
73BXD38F69R0729F1Feb05UMemphis
74BXD38M69R0731F1Jan04UMemphis
75BXD39F76R1712F1Feb05JAX
76BXD39M71R0602F1Feb05UAB
77BXD40F184R0741F1Feb05UAB
78BXD40M56R0894F1Feb05UMemphis
79BXD42F100R0742F1Feb05UAB
80BXD42M97R0936F1Jan04UMemphis
81BXD42M105R0937F1Feb05UMemphis
82BXD43M63R1047F1Feb05UTM RW
83BXD44F57R1069F1Feb05UTM RW
84BXD44M58R1072F1Feb05UTM RW
85BXD45F58R1398F1Feb05UTM RW
86BXD48F59R0946F1Feb05UTM RW
87BXD48M64R0970F1Feb05UTM RW
88BXD51F63R1430F1Feb05UTM RW
89BXD51M65R1001F1Feb05UTM RW
90BXD60F64R0976F1Feb05UTM RW
91BXD60M59R1075F1Feb05UTM RW
92BXD62F59R1033F1Feb05UTM RW
93BXD62M58R1027F1Feb05UTM RW
94BXD69F60R1438F1Feb05UTM RW
95BXD69M64R1193F1Feb05UTM RW
96BXD73F60R1275F1Feb05UTM RW
97BXD73M76R1442F1Feb05UTM RW
98BXD77M61R1426F1Feb05UTM RW
99BXD86F77R1414F1Feb05UTM RW
100BXD86M77R1418F1Feb05UTM RW
101BXD87F89R1713F1Feb05UTM RW
102BXD87M84R1709F1Feb05UTM RW
103BXD90M61R1452FFeb05UTM RW
104BXD92F58R1299F1Feb05UTM RW
105BXD92M59R1307F1Feb05UTM RW

    About the array platform :

Affymetrix Mouse Genome 430A and B array pairs: The 430A and B array pairs consist of 992936 25-nucleotide probes that collectively estimate the expression of approximately 39,000 transcripts. The array sequences were selected late in 2002 using Unigene Build 107. The arrays nominally contain the same probe sequences as the 430 2.0 series. However, we have found that roughy 75000 probes differ from those on A and B arrays and those on the 430 2.0

    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 two batches (n = 34 and n = 71 array pairs) 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 7a: The 430A and 430B arrays include a set of 100 shared probe sets (a total of 2200 probes) that have identical sequences. These probes and probe sets provide a way to calibrate expression of the 430A and 430B arrays to a common scale. To bring the two arrays into alignment, we regressed Z scores of the common set of probes to obtain a linear regression correction to rescale the 430B arrays to the 430A array. In our case this involved multiplying all 430B Z scores by the slope of the regression and adding or subtracting a small offset. The result of this step is that the mean of the 430A expression is fixed at a value of 8, whereas that of the 430B chip is typically reduced to 7. The average of the merged 430A and 430B array data set is approximately 7.5.
  • Step 7b: We recentered the merged 430A and 430B data sets to a mean of 8 and a standard deviation of 2. This involved reapplying Steps 3 through 5.
  • Step 8: 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, age, source of animals, 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). The original CEL files were read into the R environment (Ihaka and Gentleman 1996). Data were processed using the Robust Multichip Average (RMA) method (Irrizary et al. 2003). Values were log2 transformed. Probe set values listed in WebQTL are the averages of biological replicates within strain. A few technical replicates were averaged and treated as single samples. A 1-unit difference represents roughly a two-fold difference in expression level. Expression levels below 5 are usually close to background noise levels.

This data set include further normalization to produce final estimates of expression that can be compared directly to the other transforms (average of 8 units and stabilized standard deviation of 2 units within each array). Please seee Bolstad and colleagues (2003) for a helpful comparison of RMA and two other common methods of processing Affymetrix array data sets.

    About the chromosome and megabase position values:

The chromosomal locations of probe sets included on the microarrays were determined by BLAT analysis using the Mouse Genome Sequencing Consortium May 2004 Assembly (see http://genome.ucsc.edu/cgi-bin/hgBlat?command=start&org=mouse). We thank Dr. Yan Cui (UTHSC) for allowing us to use his Linux cluster to perform this analysis.

    Data source acknowledgment:

Support for acquisition of microarray data were generously provided by the NIAAA and its INIA grant program to RWW, Thomas Sutter, and Daniel Goldowitz (U01AA013515, U01AA013499-03S1, U01AA013488, U01AA013503-03S1). Support for the continued development of the GeneNetwork and WebQTL was provided by a NIMH Human Brain Project grant (P20MH062009). All arrays were processed at the University of Memphis by Thomas Sutter and colleagues with support of the INIA Bioanalytical Core.

    Information about this text file:

This text file originally generated by RWW, YHQ, and EJC, Oct 2004. Updated by RWW, Nov 5, 2004; April 7, 2005; RNA/tissue preparation protocol updatedby JLP, Sept 2, 2005; Sept 26, 2005.