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- VCU BXD PFC EtOH M430 2.0 (Dec06) RMA
- -Summary: - -This BXD data set provides estimates of mRNA expression in the prefrontal cortex following ethanol treatment across 27 BXD recombinant inbred strains and their B6 and D2 progenitor strains. All samples are from a total of 468 adult male animals obtained from Jackson Laboratory and raised in a standard laboratory environment. An average of 8 males per strain was used to measure anxiety-like behavior in response to restraint and treatment with 1.8g/kg ethanol in the light-dark transition model of anxiety. Four hours after treatment, animals were rapidly sacrificed by cervical dislocation, brains were removed, cooled and microdissected as previously described (Kerns et al., J. Neurosci. 25:2255, 2005). -All RNA isolation and subsequent probe generation and hybridization to microarrays were completed using a supervised randomization procedure to minimize batch effects. Affymetrix M430 type 2.0 microarrays were used for hybridization using standard procedures. Expression analysis was conducted by estimating the relative abundance of over 45,000 transcripts in the prefrontal cortex following ethanol treatment using the Robust Multichip Average (RMA) method. - -Animals and Tissue Used to Generate This Set of Data: - -All animals were obtained at 8-9 weeks of age from the Jackson Laboratory (Bar Harbor, ME) and were treated, behaviorally tested and brains dissected by Alex Putman and colleagues at VCU. Following an hour acclimation period to the behavioral room, animals were restrained for 15 minutes, immediately injected (I.P.) with either saline (0.9%) or 1.8g/kg ethanol, and 5 minutes later placed in the light-dark box for a 10-minute test session. All behavioral testing occurred between 10 AM and 1 PM during the light phase over a 12 month period beginning August 2005. Four hours after treatment, animals were rapidly sacrificed by cervical dislocation, brains were removed, cooled and microdissected. Prefrontal cortex tissue was isolated by microdissection using a wedge-shaped slice taken from a 4 mm thick brain slice extending rostrally from the optic chiasm. The wedge was centered on the inter-hemispheric fissure and extending 2 mm laterally on each side and ventrally to just above the corpus callosum. This tissue and all other brain regions were dissected in less than 5 minutes per mouse and were immediately frozen in liquid nitrogen followed by storage at -80 oC prior to RNA isolation. A pool of dissected tissue from 3 mice of the same strain was used to generate RNA samples. All RNA samples were extracted at VCU by Alex Putman during October 2006 and the order of RNA isolation was randomized across all strains and treatment groups (since saline treated animals were processed concurrently). - -Sample Processing: - -All samples were processed by Paul Vorster and Alex Putman at VCU between October and November 2006. The BioRad Experion RNA analyzer and used to assess total RNA integrity and verify equal molar ratios of 18S and 28S ribosomal RNA. Standard Affymetrix reagents and protocols were used for generation of cDNA and biotinylated cRNA from total RNA samples. Integrity of cRNA was checked by Experion analysis prior to microarray hybridizations. All probes exceeded a maximum size of 3000 nt for the upper border of the cRNA size distribution. - - -Replication and Sample Balance: - -At present, this ethanol prefrontal cortex mRNA expression BXD data set is represented by a total of 1 microarray for each BXD strain and 3 microarrays for each progenitor strain. - -Experimental Design and Batch Structure: - -This data set was generated concurrently with the VCU saline prefrontal cortex BXD RMA data and therefore consisted of 64 microarrays processed in 5 groups of 8 to 16 microarrays during the month of September 2006. All RNA extractions, cRNA synthesis, and hybridizations were randomized across strain and treatment groups to minimize batch effects. - - References: - Many of the techniques used to generate this data set are described in a recent publication in the Journal of Neuroscience. - - - - -Data Source Acknowledgments: - -Data were generated with funds to Mike Miles from the NIAAA. - - - |
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