Summary:
This BXD data set provides estimates of ventral tegmental area (VTA) mRNA expression in response to ethanol (1.8 g/kg x 4 hours) across 35 BXD recombinant inbred strains and their B6 and D2 progenitor strains. All samples are from a total of 596 adult male animals obtained from Jackson Laboratory (27 “classical” BXD strains) or Oak Ridge National Laboratory (extended BXD series) 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 ethanol (IP) treatment in the light-dark transition model of anxiety.
Behavior was measured for 10 minutes and then mice returned to their home cages. 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 VTA in response to ethanol 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 0.9% saline 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 16 month period beginning in August 2005. Four hours after treatment, animals were rapidly sacrificed by cervical dislocation, brains were removed, cooled and microdissected. Ventral tegmental area tissue was isolated by microdissection using a wedge-shaped slice as described in Kerns et al., 2005. 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 Nate Bruce during April 2009 and the order of RNA isolation was randomized across all strains and treatment groups (since ethanol treated animals were processed concurrently).
Sample Processing:
All samples were processed by Nate Bruce at VCU between April and May 2009. The BioRad Experion RNA analyzer and used to assess total RNA integrity and verify equal molar ratios of 18S and 28S ribosomal RNA. All RNA Quality Index (RQI) calculations were > 8. 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 VTA mRNA expression BXD data set is represented by a total of 1 microarray for each BXD strain and 5 microarrays for each progenitor strain. A duplicate dataset for BXD strains is in progress.
Experimental Design and Batch Structure:
This data set was generated concurrently with the VCU saline VTA BXD RMA data and therefore consisted of 90 microarrays processed in 6 groups of 8 to 16 microarrays during the month of May 2009. All RNA extractions, cRNA synthesis, and hybridizations were randomized across strain and treatment groups to minimize batch effects.
Data Source Acknowledgements:
Data were generated with NIAAA grants U01AA016662, U01AA01667 and R01AA014717 to Michael F. Miles. BXD mice obtained from Oak Ridge National Laboratory were through the Mouse Research Core of the Interactive Neuroscience Initiative on Alcoholism – Stress (INIA-Stress) consortium. Assistance for this work from INIA-Stress investigators Elissa Chesler, Dan Goldowitz, Lu Lu and Robert Williams was greatly appreciated.
