Computational protocol: Age-related molecular genetic changes of murine bone marrow mesenchymal stem cells

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Protocol publication

[…] Equal amounts of RNA from 4 animals per age group were combined to generate a pooled sample for each experimental group. The pooled RNA samples were labeled for hybridization to the Affymetrix Mouse Genome 430 2.0 GeneChip Array, using standard Affymetrix protocols. This chip contains roughly 39,000 transcripts. A total of 11 chips were run including triplicates of 2-month samples and quadruplicates of 8 month and 12 month samples. Arrays were pre-hybridized with 1× Hybridization Buffer for 10 minutes at 45°C. The labeled samples were added to the GeneChip Arrays and hybridized for 16 hours at 45°C. The arrays were stained and washed according to Affymetrix Fluidics Station 450 protocol EukGEWS2v5_450. The intensity values were collected from the GeneChips by scanning the arrays with a GeneChip Scanner 3000 7G. The resultant images were analyzed with the MAS5 algorithm for quality control checks. Pearson correlation coefficients taken from plotting signal intensity values of duplicate chips across all genes validated that triplicate experiments were similar (typical correlation coefficients for previous double amplification experiments have been r2 ~.988). Chip intensity values were calculated using the gcrma algorithm. Chips were normalized with the quantile normalization procedure. Normalized expression values from the raw data were generated using default settings for the GC-Robust Multi-array Average (GC-RMA) method that provides the best balance of accuracy and precision [] within GeneSpring (Silicon Genetics, Redwood City, CA). Subsequent statistical analysis was also performed in GeneSpring. The cross-gene error model was applied with replicates. The acceptance criterion for gene array expression changes was a minimum fold change of 2.0 and a t-test p-value of < 0.05. Venn diagrams and scatter plots were generated within GeneSpring. […]

Pipeline specifications

Software tools GC–RMA, GeneSpring GX
Application Gene expression microarray analysis
Organisms Mus musculus