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Summary
This August 2005 data freeze provides estimates of mRNA expression in brains of BXD recombinant inbred mice measured using Affymetrix U74Av2 microarrays. Data were generated at the University of Tennessee Health Science Center (UTHSC). Over 300 brain samples from 35 strains were hybridized in small pools (n=3) to 97 arrays. Data were processed using the Microarray Suite 5 (MAS 5) protocol of Affymetrix. To simplify comparison between transforms, MAS 5 values of each array were log2 transformed and adjusted to an average of 8 units. In general, MAS 5 data do not perform as well as RMA, PDNN, or the new heritability weighted transforms (HW1PM).
About cases
This data set includes estimate of gene expression for 35 genetically uniform lines of mice: C57BL/6J (B6, or simply B), DBA/2J (D2 or D), their B6D2 F1 intercross, and 32 BXD recombinant inbred (RI) strains derived by crossing female B6 mice with male D2 mice and then inbreeding progeny for over 21 generations. This set of RI strains is a remarkable resource because many of these strains have been extensively phenotyped for hundreds of interesting traits over a 25-year period. A significant advantage of this RI set is that the two parental strains (B6 and D2) have both been extensively sequenced and are known to differ at approximately 1.8 million SNPs. Coding variants (mostly single nucleotide polymorphisms and insertion-deletions) that may produce interesting phenotypes can be rapidly identified in this particular RI set.
BXD1 through BXD32 were produced by Benjamin A. Taylor starting in the late 1970s. BXD33 through BXD42 were also produced by Taylor, but from a second set of crosses initiated in the early 1990s. These strains are all available from the Jackson Laboratory, Bar Harbor, Maine. BXD43 through BXD99 were produced 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). Only two of these incipient strains are included in the current database (BXD67 and BXD68).
In this mRNA expression database we generally used progeny of stock obtained from The Jackson Laboratory between 1999 and 2001. Animals were generated in-house at the University of Alabama by John Mountz and Hui-Chen Hsu and at the University of Tennessee Health Science Center by Lu Lu and Robert Williams.
The table below lists the arrays by strain, sex, and age. Each array was hybridized to a pool of mRNA from three mice.
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About tissue
Each array was hybridized with labeled cRNA generated from a pool of three brains from adult animals usually of the same age and always of the same sex. The brain region included most of the forebrain and midbrain, bilaterally. However, the sample excluded the olfactory bulbs, retinas, or the posterior pituitary (all formally part of the forebrain). A total of 97 such pooled samples were arrayed: 73 from females and 24 from males. Animals ranged in age from 56 to 441 days, usually with a balanced design: one pool at approximately 8 weeks, one pool at approximately 20 weeks, and one pool at approximately 1 year. Strain averages of mRNA expression level are therefore typically based on three pooled biological replicate arrays. This data set does not incorporate statistical adjustment for possible effects of age and sex. Users can select the strain symbol in the table above to review details about the specific cases and array processing center (DP = Divyen Patel at Genome Explorations, Inc; TS = Thomas Sutter at University of Memphis). You can also click on the individual symbols (males or females) to view the array image.
About platform
Affymetrix U74Av2 GeneChip: The expression data were generated using 97 U74Av2 arrays. The chromosomal locations of U74Av2 probe sets were determined by BLAT analysis of concatenated probe sequences using the Mouse Genome Sequencing Consortium Mar 2005 (mm6) assembly. This BLAT analysis is performed periodically by Yanhua Qu as each new build of the mouse genome is released (see http://genome.ucsc.edu/cgi-bin/hgBlat?command=start&org=mouse). We thank Yan Cui (UTHSC) for allowing us to use his Linux cluster to perform this analysis. It is possiible to confirm the BLAT alignment results yourself simply by clicking on the Verify link in the Trait Data and Editing Form (right side of the Location line).
Most probe sets on the U74Av2 array consist of a total of 32 probes, divided into 16 perfect match probes and 16 mismatch controls. Each set of these 25-nucleotide-long probes has an identifier code that includes a unique number, an underscore character, and several suffix characters that highlight design features. The most common probe set suffix is at. This code indicates that the probes should hybridize relatively selectively with the complementary anti-sense target (i.e., the complemenary RNA) produced from a single gene. Other codes include:
- f_at (sequence family): Some probes in this probe set will hybridize to identical and/or slightly different sequences of related gene transcripts.
- s_at (similarity constraint): All Probes in this probe set target common sequences found in transcripts from several genes.
- g_at (common groups): Some probes in this set target identical sequences in multiple genes and some target unique sequences in the intended target gene.
- r_at (rules dropped): Probe sets for which it was not possible to pick a full set of unique probes using the Affymetrix probe selection rules. Probes were picked after dropping some of the selection rules.
- i_at (incomplete): Designates probe sets for which there are fewer than the standard numbers of unique probes specified in the design (16 perfect match for the U74Av2).
- st (sense target) : Designates a sense target; almost always generated in error.
Descriptions for the probe set extensions were taken from the Affymetrix GeneChip Expression Analysis Fundamentals.
About data processing
Probe (cell) level data from the CEL file: Probe signal intensity estimates in the Affymetrix CEL files are the 75% quantile value taken from a set of 36 (6x6) pixels per probe cell in the DAT image file.Probe set data from the CHP file: Probe set estimates of expression were initially generated using the standard Affymetrix MAS 5 algorithm. The CHP values were then processed following precisely the same six steps listed above to normalize expression and stabilize the variance of all 97 arrays. The mean expression within each array is therefore 8 units with a standard deviation of 2 units. A 1-unit difference represents roughly a 2-fold difference in expression level. Expression levels below 5 are close to the background noise level. While a value of 8 unit is nominally the average expression, this average includes all those transcripts with negligible expression in the brain that would often be eliminated from subsequent analysis (so-called "absent" and "marginal" calls in the CHP file).
- 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 for the log base 2 values for the total set of 97 arrays (all seven 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 corrected for technical variance introduced by seven batches at the probe level. To do this we determined the ratio of the batch mean to the mean of all seven 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 of the seven batches is the same.
- Step 7: Finally, we computed the arithmetic mean of the values for the set of microarrays for each strain. Technical replciates 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 these variables.
Acknowledgment
Data were generated with funds to RWW from the Dunavant Chair of Excellence, University of Tennessee Health Science Center, Department of Pediatrics. The majority of arrays were processed at Genome Explorations by Dr. Divyen Patel. We thank Guomin Zhou for generating advanced intercross stock used to produce most of the new BXD RI strains.
Notes
This text file originally generated by RWW, YHQ, and EJC, December 2003. Updated by RWW, October 29, 2004.