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VCU BXD PFC CIE EtOH M430 2.0 (Jan11) RMA **
Accession number: GN300 
Summary:
This BXD data set provides estimates of prefrontal cortex (PFC) mRNA expression across 25 BXD RI strains along with their B6 and D2 progenitor strains and F1 strain. Mice were exposed to an established dependence and relapse drinking model (e.g., Becker and Lopez, 2004; Lopez and Becker, 2005; Griffin et al., 2009). Briefly, a 2-bottle choice (15% v/v ethanol vs. water) limited access (2 hr/day) drinking model was employed. After 6 weeks of establishing baseline intake, mice from each genotype received 4 weekly cycles of chronic intermittent ethanol (CIE) vapor exposure (EtOH group) or air exposure (CTL group) in inhalation chambers (16 hr/day x 4 days + 72 hr forced abstinence) alternated with weekly test cycles in which ethanol intake was measured during 5 consecutive limited access daily drinking sessions. Mice were not food or water deprived at any time during the study. This study was conducted as the first part of an overall design, with the second part to be completed using complementary male and female mice of the corresponding genotypes used in the present study. The general study design involved typically one mouse per experimental cell (N= 1/genotype/sex/group) with both the CTL and EtOH samples of a given strain being of the same sex. A positive control condition (C57BL/6J male mice) was included in the study (N= 6-8/group). All mice received a 5th CIE exposure cycle and EtOH and CTL groups were sacrificed at 72 hr following removal from the inhalation chambers. Male animals were used for strains BXD5, BXD12, BXD14, BXD16, BXD34, BXD39, BXD43, BXD45, BXD66, BXD74, BXD77, BXD80, BXD81, BXD83, BXD100, BXD101, BXD102 and BXD103 while females were used for BXD49, BXD50, BXD55, BXD62, BXD71, BXD75 and BXD85
All RNA isolation and subsequent probe generation and hybridization to microarrays were completed using a supervised randomization procedure to minimize batch effects. Affymetrix Mouse Genome 430 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 PFC using the Robust Multichip Average (RMA) method. Comparison of air vs. ethanol vapor treated animals (within strain comparisons) were also done at the individual probe level by using the S-score analysis algorithm developed in the Miles's laboratory (Zhang et all, 2002; Kennedy et al., 2006). Three datasets were deposited within GeneNetwork: VCU_BXD_PFC_CIE_Air M430 2.0 (12/10) RMA (RMA values of arrays from air-treated controls); VCU_BXD_PFC_CIE_EtOHVapor M430 2.0 (12/10) RMA (RMA values from ethanol-vapor treated animals); and VCU_BXD_PFC_CIE_Sscore M430 2.0 (12/10) RMA (S-score comparison of ethanol vapor vs. air control arrays within strains).
About the animals and tissue used to generate this set of data:
All 121 mice (85 males and 36 females) were obtained from R. Williams's lab. Immediately after being sacrificed, brains were removed, cooled and microdissected as previously described (Kerns et al., J. Neurosci. 25:2255, 2005). Prefrontal cortex tissue was isolated by microdissection using a wedge-shaped slice as described in Kerns et al., 2005. This tissue was immediately frozen in liquid nitrogen followed by storage at -80 oC prior to RNA isolation.
Sample Processing:
All RNA samples were extracted at VCU by Paul Vorster during December 2010. The order of RNA isolation was randomized across all strains and treatment groups (since ethanol treated animals were processed concurrently). The BioRad Experion RNA analyzer was 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:
This study was conducted as the first part of an overall design, with the second part to be completed using complementary male and female mice of the corresponding genotypes used in the present study. The general study design involved typically one mouse per experimental cell (N= 1/genotype/sex/group). At present, this ethanol vapor PFC mRNA expression data set is represented by 1-2 microarrays for all strains, except the C57BL/6J male mice, which were included as a positive control condition and were represented by 6-8 microarrays. In cases where there multiple arrays run per strain/treatment group, an average of RMA results is reported. Similarly, S-scores were calculated in such cases by first averaging .Cel files across biological replicates.
Experimental Design and Batch Structure:
This data consisted of 70 microarrays processed in 9 batches of 8 arrays during the month of December 2010. All RNA extractions, cRNA synthesis, and hybridizations were randomized across strain and treatment groups to minimize batch effects.
| Sample | RNA Batch | cRNA Synth | Array Scan Batch |
| II3M_B6J_C | 5 | 6 | 4 |
| II6M_B6J_C | 3 | 5 | 1 |
| II9M_B6J_C | 9 | 3 | 7 |
| II10M_B6J_C | 2 | 4 | 5 |
| II12M_B6J_C | 8 | 1 | 2 |
| II13M_B6J_C | 4 | 6 | 6 |
| II15M_B6J_C | 7 | 1 | 8 |
| II16M_B6J_C | 1 | 4 | 3 |
| II2M_B6J_E | 3 | 5 | 3 |
| II4M_B6J_E | 2 | 6 | 4 |
| II7M_B6J_E | 5 | 2 | 6 |
| II8M_B6J_E | 1 | 4 | 7 |
| II11M_B6J_E | 6 | 2 | 8 |
| II14M_B6J_E | 4 | 1 | 5 |
| H2M_B6UT_C | 6 | 3 | 7 |
| H1M_B6UT_E | 3 | 6 | 5 |
| R2M_BXD5_C | 1 | 2 | 3 |
| R1M_BXD5_E | 2 | 5 | 1 |
| U2M_BXD12_C | 2 | 5 | 6 |
| U1M_BXD12_E | 9 | 3 | 7 |
| G2M_BXD14_C | 8 | 1 | 7 |
| G1M_BXD14_E | 5 | 5 | 9 |
| K2M_BXD16_C | 3 | 6 | 5 |
| K1M_BXD16_E | 1 | 4 | 8 |
| F2M_BXD34_C | 2 | 2 | 2 |
| F1M_BXD34_E | 4 | 1 | 4 |
| BB1M_BXD39_E | 1 | 3 | 9 |
| BB2M_BXD39_E | 7 | 6 | 2 |
| Z3M_BXD43_C | 5 | 2 | 6 |
| Z2M_BXD43_E | 7 | 3 | 5 |
| L2M_BXD45_C | 1 | 3 | 7 |
| L1M_BXD45_E | 6 | 4 | 1 |
| A2M_BXD66_C | 4 | 5 | 6 |
| A1M_BXD66_E | 9 | 4 | 3 |
| B2M_BXD74_C | 9 | 2 | 8 |
| DD2M_BXD77_C | 7 | 1 | 5 |
| DD1M_BXD77_E | 6 | 4 | 4 |
| C3M_BXD80_C | 5 | 5 | 1 |
| C4M_BXD80_E | 2 | 6 | 8 |
| C5M_BXD80_E | 9 | 3 | 6 |
| J2M_BXD81_C | 8 | 1 | 2 |
| J1M_BXD81_E | 4 | 2 | 3 |
| Y2M_BXD83_C | 3 | 4 | 9 |
| Y1M_BXD83_E | 9 | 1 | 2 |
| N2M_BXD100_C | 8 | 3 | 3 |
| N1M_BXD100_E | 4 | 5 | 6 |
| E2M_BXD101_C | 7 | 4 | 1 |
| E1M_BXD101_E | 6 | 1 | 2 |
| D1M_BXD102_E | 8 | 2 | 2 |
| HH2M_BXD103_C | 6 | 2 | 8 |
| HH1M_BXD103_E | 3 | 5 | 7 |
| A2F_D2B6F1_C | 6 | 3 | 6 |
| A1F_D2B6F1_E | 8 | 1 | 3 |
| B2F_D2_C | 7 | 1 | 7 |
| Q2F_BXD49_C | 2 | 2 | 3 |
| Q3F_BXD49_E | 9 | 3 | 9 |
| K2F_BXD50_C | 4 | 4 | 1 |
| K1F_BXD50_E | 8 | 6 | 8 |
| G1F_BXD55_C | 1 | 5 | 9 |
| G2F_BXD55_E | 2 | 4 | 7 |
| G3F_BXD55_E | 5 | 1 | 4 |
| N2F_BXD62_C | 5 | 5 | 2 |
| N1F_BXD62_E | 6 | 2 | 6 |
| D2F_BXD71_C | 5 | 3 | 5 |
| D1F_BXD71_E | 4 | 6 | 2 |
| M2F_BXD75_C | 3 | 5 | 4 |
| M1F_BXD75_E | 1 | 3 | 9 |
| M3F_BXD75_E | 7 | 4 | 5 |
| J2F_BXD85_E | 7 | 3 | 8 |
| J3F_BXD85_E | 3 | 6 | 1 |
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Data source acknowledgment:
Any use of this data in publications should come with the approval of Dr. Michael Miles (mfmiles@vcu.edu) at the present time since the data is unpublished. Use of the this data is available as a collaborative project until this data has been included in a primary publication. Upon use of this data, acknowledgement should be given to Drs. Michael Miles, Robert Williams and Howard Becker, whose laboratories collaborated in the generation of this dataset. Funding acknowledgments should include NIAAA grants U01 AA016667 and U01 AA016662 to MFM.
Information about this text file:
This file last updated by A.Centeno on 1-25-2011
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