SJUT Cerebellum mRNA M430 (Mar05) RMA

Accession number: GN56

    Summary:

This March 2005 data freeze provides estimates of mRNA expression in adult cerebellum of 48 lines of mice including 45 BXD recombinant inbred strains, C57BL/6J, DBA/2J, and F1 hybrids. Data were generated by a consortium of investigators at St. Jude Children's Research Hospital (SJ) and the University of Tennessee Health Science Center (UT). Cerebellar samples were hybridized in small pools (n = 3) to Affymetrix M430A and B arrays. Data were processed using the RMA protocol. To simplify comparison between transforms, PDNN 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 exploited a set of BXD recombinant inbred strains. All BXD lines are derived crossed between C57BL/6J (B6 or B) and DBA/2J (D2 or D). Both B and D parental strains have been almost fully sequenced (8x coverage for B6 by a public consortium and approximately 1.5x coverage for D by Celera Discovery Systems) and data for 1.75 millioin B vs D SNPs are incorporated into WebQTLs genetic maps for the BXDs. BXD2 through BXD32 were produced by Benjamin A. Taylor starting in the late 1970s. BXD33 through 42 were also produced by Taylor, but they were generated in the 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).

Most BXD animals were generated in-house at the University of Tennessee Health Science Center by Lu Lu and Robert Williams using stock obtained from The Jackson Laboratory between 1999 and 2004. All BXD strains with numbers above 42 are new advanced intecross type BXDs (Peirce et al. 2004) that are current available from UTHSC. Additional cases were provided by Glenn Rosen, John Mountz, and Hui-Chen Hsu. These cases were bred either at The Jackson Laboratory (GR) or at the University of Alabama (JM and HCH).

    About the tissue used to generate this set of data:

The March 2005 data set consists of a total of 102 array pairs (Affymetrix 430A and 430B) from 49 different genotypes. Each sample consists of whole cerebellum taken from three adult animals of the same age and sex. Two sets of technical replicates (BXD14 n = 2; BXD29 n = 3) were combined before generating group means; giving a total of 101 biologically independent data sets. The two reciprocal F1s (D2B6F1 and B6D2F1) were combined to give a single F1 mean estimate of gene expression. 430A and 430B arrays were processed in three large batches. The first batch (May03 data) consists of 17 samples from 17 strains balanced by sex (8M and 9F). The second batch consists of 29 samples, and includes biological replicates, 2 technical replicates, and data for 9 new strains. The third batch consists of 56 samples, and also includes biological replicates, 2 technical replicates, and data for 15 additional strains.

Replication and Sample Balance: Our goal is to obtain data for independent biological sample pools from both sexes for each strain. Six of 48 genotypes are still represented by single samples: BXD13, BXD20, BXD27 are female-only strains, whereas BXD25, BXD77, BXD90 are male-only. Ten strains are represented by three independent samples with the following breakdown by sex: C57BL/6J (1F 2M), DBA/2J (2F 1M), B6D2F1 (1F 2M), BXD2 (2F 1M), BXD11 (2F 1M), BXD28 (2F 1M), BXD40 (2F 1M), BXD51 (1F 2M), BXD60 (1F 2M), BXD92 (2F 1M).

The age range of samples is relatively narrow. Only 18 samples were taken from animals older than 99 days and only two samples are older than 7 months of age. BXD11 includes an extra (third) 441-day-old female sample and the BXD28 includes an extra 427-day-old sample.

RNA was extracted at UTHSC by Lu Lu, Zhiping Jia, and Hongtao Zhai.

All samples were subsequently processed at the Hartwell Center Affymetrix laboratory at SJCRH by Jay Morris.

The table below summarizes informaton on strain, sex, age, sample name, and batch number.

Id Strain Sex Age SampleName BatchID Source 1 C57BL/6J F 116 R0773C 2 UAB 2 C57BL/6J M 109 R0054C 1 JAX 3 C57BL/6J M 71 R1450C 3 UTM DG 4 DBA/2J F 71 R0175C 1 UAB 5 DBA/2J F 91 R0782C 2 UAB 6 DBA/2J M 62 R1121C 3 UTM RW 7 B6D2F1 F 60 R1115C 3 UTM RW 8 B6D2F1 M 94 R0347C 1 JAX 9 B6D2F1 M 127 R0766C 2 UTM JB 10 D2B6F1 F 57 R1067C 3 UTM RW 11 D2B6F1 M 60 R1387C 3 UTM RW 12 BXD1 F 57 R0813C 2 UAB 13 BXD1 M 181 R1151C 3 UTM JB 14 BXD2 F 142 R0751C 1 UAB 15 BXD2 F 78 R0774C 2 UAB 16 BXD2 M 61 R1503C 3 HarvardU GR 17 BXD5 F 56 R0802C 2 UMemphis 18 BXD6 F 92 R0719C 1 UMemphis 19 BXD6 M 92 R0720C 3 UMemphis 20 BXD8 F 72 R0173C 1 UAB 21 BXD8 M 59 R1484C 3 HarvardU GR 22 BXD9 F 86 R0736C 3 UMemphis 23 BXD9 M 86 R0737C 1 UMemphis 24 BXD11 F 441 R0200C 1 UAB 25 BXD11 F 97 R0791C 3 UAB 26 BXD11 M 92 R0790C 2 UMemphis 27 BXD12 F 130 R0776C 2 UAB 28 BXD12 M 64 R0756C 2 UMemphis 29 BXD13 F 86 R1144C 3 UMemphis 30 BXD14 F 190 R0794C 2 UAB 31 BXD14 F 190 R0794C 3 UAB 32 BXD14 M 91 R0758C 2 UMemphis 33 BXD14 M 65 R1130C 3 UTM RW 34 BXD15 F 60 R1491C 3 HarvardU GR 35 BXD15 M 61 R1499C 3 HarvardU GR 36 BXD16 F 163 R0750C 1 UAB 37 BXD16 M 61 R1572C 3 HarvardU GR 38 BXD19 F 61 R0772C 2 UAB 39 BXD19 M 157 R1230C 3 UTM JB 40 BXD20 F 59 R1488C 3 HarvardU GR 41 BXD21 F 116 R0711C 1 UAB 42 BXD21 M 64 R0803C 2 UMemphis 43 BXD22 F 65 R0174C 1 UAB 44 BXD22 M 59 R1489C 3 HarvardU GR 45 BXD23 F 88 R0814C 2 UAB 46 BXD24 F 71 R0805C 2 UMemphis 47 BXD24 M 71 R0759C 2 UMemphis 48 BXD25 M 90 R0429C 1 UTM RW 49 BXD27 F 60 R1496C 3 HarvardU GR 50 BXD28 F 113 R0785C 2 UTM RW 51 BXD28 M 79 R0739C 3 UMemphis 52 BXD29 F 82 R0777C 2 UAB 53 BXD29 M 76 R0714C 1 UMemphis 54 BXD29 M 76 R0714C 2 UMemphis 55 BXD29 M 76 R0714C 3 UMemphis 56 BXD31 F 142 R0816C 2 UAB 57 BXD31 M 61 R1142C 3 UTM RW 58 BXD32 F 62 R0778C 2 UAB 59 BXD32 M 218 R0786C 2 UAB 60 BXD33 F 184 R0793C 2 UAB 61 BXD33 M 124 R0715C 1 UAB 62 BXD34 F 56 R0725C 1 UMemphis 63 BXD34 M 91 R0789C 2 UMemphis 64 BXD36 F 64 R1667C 3 UTM RW 65 BXD36 M 61 R1212C 3 UMemphis 66 BXD38 F 55 R0781C 2 UAB 67 BXD38 M 65 R0761C 2 UMemphis 68 BXD39 F 59 R1490C 3 HarvardU GR 69 BXD39 M 165 R0723C 1 UAB 70 BXD40 F 56 R0718C 2 UMemphis 71 BXD40 M 73 R0812C 2 UMemphis 72 BXD42 F 100 R0799C 2 UAB 73 BXD42 M 97 R0709C 1 UMemphis 74 BXD43 F 61 R1200C 3 UTM RW 75 BXD43 M 63 R1182C 3 UTM RW 76 BXD44 F 61 R1188C 3 UTM RW 77 BXD44 M 58 R1073C 3 UTM RW 78 BXD45 F 63 R1404C 3 UTM RW 79 BXD45 M 93 R1506C 3 UTM RW 80 BXD48 F 64 R1158C 3 UTM RW 81 BXD48 M 65 R1165C 3 UTM RW 82 BXD51 F 66 R1666C 3 UTM RW 83 BXD51 M 62 R1180C 3 UTM RW 84 BXD51 M 79 R1671C 3 UTM RW 85 BXD60 F 64 R1160C 3 UTM RW 86 BXD60 M 61 R1103C 3 UTM RW 87 BXD60 M 99 R1669C 3 UTM RW 88 BXD62 M 61 R1149C 3 UTM RW 89 BXD62 M 60 R1668C 3 UTM RW 90 BXD69 F 60 R1440C 3 UTM RW 91 BXD69 M 64 R1197C 3 UTM RW 92 BXD73 F 60 R1276C 3 UTM RW 93 BXD73 M 77 R1665C 3 UTM RW 94 BXD77 M 62 R1424C 3 UTM RW 95 BXD85 F 79 R1486C 3 UTM RW 96 BXD85 M 79 R1487C 3 UTM RW 97 BXD86 F 58 R1408C 3 UTM RW 98 BXD86 M 58 R1412C 3 UTM RW 99 BXD90 M 74 R1664C 3 UTM RW 100 BXD92 F 62 R1391C 3 UTM RW 101 BXD92 F 63 R1670C 3 UTM RW 102 BXD92 M 59 R1308C 3 UTM RW

    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 for the log base 2 values for the total set of 104 arrays (all three 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 three large batches at the probe level. To do this we determined the ratio of the batch mean to the mean of all three 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 three batches 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 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.

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 and gene markers on the 430A and 430B 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:

Data were generated with funds contributed equally by The UTHSC-SJCRH Cerebellum Transcriptome Profiling Consortium. Our members include:

• Tom Curran
• Dan Goldowitz
• Kristin Hamre
• Lu Lu
• Peter McKinnon
• Jim Morgan
• Clayton Naeve
• Richard Smeyne
• Robert Williams
• The Center of Genomics and Bioinformatics at UTHSC
• The Hartwell Center at SJCRH

    Information about this text file:

This text file originally generated by RWW and YHQ, March 21, 2005. Updated by RWW, March 23, 2005.