Authors: Joshua Hunsberger & Samuel Newton
Below are the procedures we used for microarray analysis.
- Isolate total hippocampal RNA from individual animals using (RNA Aqueous, Ambion).
- Determine RNA quality by measuring optical density values (260/280). Values should be consistently at or over 1.9.
- Use five micrograms of total RNA from experimental and control animals (e.g. 1 week exercise vs. sedentary mice) and reverse-transcribe into cDNA.
- Indirectly label using a sensitive fluorescent labeling procedure (Genisphere).
- A two-step hybridization and labeling protocol was used where the chip was hybridized to cDNA overnight, washed stringently to remove nonspecifically bound probe, and then poststained with fluorescent dendrimers.
- After posthybridization and washes, scan slides using a GenePix scanner (Axon Instruments). Image analysis was performed using GenePix Pro 4.0 software.
- Import the resulting files from GenePix 4.0 (Axon Instruments) analysis into Genespring 5.0 (Silicon Genetics) for additional visualization and data mining.
- Only consider hybridization spots as positive if there is a signal intensity of twice the background or more in at least one channel of half of the replicates.
- Perform per-chip normalization by dividing the expressed genes by the median of two housekeeping control genes, β-tubulin and cyclophilin, that were not regulated.
- Determine gene regulation by taking the log ratio of the median experimental (running) channel signal to the median control (sedentary) channel signal.
- Define up-regulated genes as having an average expression ratio of >1.3, and define the down-regulated genes as having an average expression ratio of <0.7. Determine these values by performing homotypic hybridizations where the same sample is hybridized in both channels (cy3 & cy5).
- These cutoff levels are also consistent with expected levels of gene regulation in brain tissue relative to cultured cell reports by others (1,2).
- Perform statistical analysis by an unpaired t test using the cross-gene pooled error method in, for example, Genespring software. Significance was set at p < 0.05.
- Classify significantly regulated genes into relevant functional categories.
- Mirnics, K. & Pevsner, J. Progress in the use of microarray technology to study the neurobiology of disease. Nat Neurosci 7, 434-9 (2004).
- Lei, H., Wang, H., Juan, A. H. & Ruddle, F. H. The identification of Hoxc8 target genes. Proc Natl Acad Sci U S A 102, 2420-4 (2005).
Antidepressant actions of the exercise-regulated gene VGF, doi:doi:10.1038/nm1669
Joshua Hunsberger & Samuel Newton, Yale University
Source: Protocol Exchange (2007) doi:10.1038/nprot.2007.509. Originally published online 12 December 2007.