Summary:
Spleen mRNA expression levels are measured for 77 individual BXD RI mice from 24 different strains. The expressed gene set were characterised using the Affymetrix Mouse430_2.0 GeneChip which encompass over 34,000 known genes.
Animals and Tissue Used to Generate This Set of Data:
Female BXD mice were harvested between 8 and 12 weeks of age. The oestrus cycle of each mouse was determined by observing the status of the cells obtained from a vaginal swab by light microscopy. Mice were culled by cervical dislocation; the brain and spleen were harvested immediately and snap frozen on dry ice. Tissues were subsequently stored at -80°C. Individual mice were identified by strain, age and cage number and were assigned a unique sample identifier number at this stage.
RNA Extraction:
The spleens (average weight 0.1g) were homogenised individually in 1ml of TRIzol reagent per 100mg of tissue using a polytron homogenizer and a glass pestle and mortar. The polytron homogenizer was found to most quickly and efficiently disrupt the tough splenic tissue, giving rise to moderate yields of RNA of good quality with little contamination.
Homogenates were chloroform extracted using 0.2ml of chloroform per 1ml of TRIzol. These were shaken vigorously by hand and separated with the aid of phase lock heavy tubes. 0.5ml of isopropanol per 1ml of TRIzol was added to retained aqueous phase at room temperature. This was then centrifuged at 4,000 x g for 30 minutes at 2-8°C. The pellet was washed with at least 1ml of 75% ethanol per 1ml of TRIzol used, and mixed by vortexing until the pellet came loose from the tube wall. This was then centrifuged at 4,000 x g for 10 minutes at 2-8°C. The pellet was air dried, dissolved in 100μl of RNase-free water and incubated at 55-60°C for 10 minutes. The RNA sample purity and concentration was determined by gel electrophoresis and spectrophotometry
About the array platform:
The Affymetrix microarrays used in this investigation were the GeneChip ® Mouse Genome 430 2.0 Array which enables genome-wide expression analysis on a single array. These probe arrays contain over 45,000 probe sets which analyse the expression of over 39,000 transcripts and variants from over 34,000 well characterized mouse genes. Multiple probe pairs per probe set provide several independent measurements for every transcript, increasing accuracy and reproducibility. The probe sets were selected from sequences derived from GenBank®, dbEST, and RefSeq. The sequence clusters were created from the UniGene database and then refined by analysis and comparison with the publicly available draft assembly of the mouse genome from the Whitehead Institute Centre for genome Research.
eQTL Statistics:
This data set generates eQTLs with peak LRS scores of about 80 (see 1458092_at, Gene Symbol: Ap3m1). This is an impressive value given the sample size consists of only 23 BXD strains. A total of 194 probe sets are associated with LOD > 10 or LRS >46.
Researchers:
Sarah Lawn under the supervision of Cathy Fernandes, Leo Schalkwyk and Steve Whatley.
Publications:
Davies, M.N., Lawn, S., Whatley, S., Fernandes, C., Williams, R.W., Schalkwyk, L.C. (2009). , Is blood a reasonable surrogate for brain in gene expression studies? Frontiers in Neurogenomics.
