Mice were given a one-week acclimation period in combined housing and another week to acclimate to the bottle positions in individual housing. Food and water were provided ad libitum and monitored daily, as were the temperature and light/dark cycles. Mice underwent a 30-day two-bottle choice paradigm with continuous (24-hour) access to one bottle of 20% ethanol and one bottle of water (Blednov, Mayfield et al. 2012). A control group of mice received two bottles of water. Bottle weights were recorded daily, mice were weighed every four days and the amount of alcohol consumed was calculated as g/kg. Ethanol bottle positions were alternated daily to control for position preferences. All procedures were approved by the Institutional Animal Care and Use Committee and adhere to NIH Guidelines for the ethical care and use of animals in research.
Growth protocol
Adult (two month old) C57BL/6J female mice were maintained at The University of Texas at Austin Animal Resources Center.
Extracted molecule
total RNA
Extraction protocol
After 30 days of the drinking paradigm, eight alcohol mice and 13 control mice were euthanized by cervical dislocation and then decapitated. Brains were removed and washed for one minute with 1ml of ice-cold Homogenizing Buffer (HB) containing 20mM Hepes, 1mM EDTA (pH 7.4), 40 U/ml RNAseOut (Invitrogen, CA), phosphatase inhibitor cocktail 3 (Sigma, MO) and protease inhibitors “Complete” (Roche, IN). Brains were then placed in a coronal Zivic mouse brain slicer with a 0.5mm resolution (Zivic Instruments, PA) and sliced in the following coordinates in order to isolate extended amygdala: coronal level 56-66 [Bregma (-0.18)-(-1.155)] and 66-80 [Bregma (-1.155)-(-2.55)]. The extended amygdala was dissected, placed in ice-cold HB (250ml) and homogenized for one minute using a VWR homogenizer and pestle (VWR, PA). To minimize homogenate loss, pestles were washed with 50ml HB after use and the wash was collected and added to the sample. Ten percent of the homogenate (30ml) was snap frozen in liquid nitrogen and stored at -80°C for subsequent RNA total homogenate (TH) analysis. Paired synaptoneurosomes (SN) were isolated from the rest of the homogenate (270ml), see Most, Ferguson et al. 2014 for detailed methodology. Briefly, homogenates were filtered through a 100um-pore filter and subsequently through a 5um-pore filter (Millipore, MA); filters were washed with HB before use for protection from RNAse. To maximize yield, the filters were washed with 50ml HB after use and the wash was collected and added to the homogenate. The homogenate was then centrifuged at 14,000g for 20 minutes at 4°C in order to pellet the cell fraction containing SN. The supernatant was removed and the pellet snap-frozen and stored at -80°C for SN RNA analysis. Microscopy was used to further characterize the SN preparation (see Most, Ferguson et al. 2014). RNA was extracted from 21 paired SN and TH samples using the Direct-Zol RNA extraction kit (Zymo, Japan) with small IC extraction columns according to the manufacturer’s instructions. The RNA was quantified using a NanoDrop1000 (Thermo Fisher Scientific Inc., IL) and assayed for quality using an Agilent 2100 Tape Station (Agilent Technologies, CA) (Figure S1a). The criteria for RNA quantity and quality were as follows: total amount of RNA>500 ng, 280/260>1.7, and RIN>6.5. We measured RIN of five of the control samples using Agilent’s Bioanalyzer Nano kit. To ensure that samples with high RINs also included high quality and quantity of microRNAs, the same five control samples were subjected to a small RNA analysis using Agilent’s Bioanalyzer Small kit. MicroRNAs comprised > 10% of the small RNA population in the samples (Figure S1b). One TH control sample did not pass RNA quality criteria and was removed from the study as well as its corresponding SN, resulting in a total of 20 paired SN and TH samples.
Label
biotin
Label protocol
Forty microRNA samples were labeled and hybridized to Affymetrix GeneChip microRNA 3.0 Arrays (Affymetrix, CA), at the University of Texas Southwestern Medical Center microarray facility in Dallas. This microarray platform uses miRBase version 17 and contains 19,724 probes for mature microRNAs from 153 organisms.
Hybridization protocol
Performed at University of Texas Southwestern Medical Center microarray facility
Scan protocol
Performed at University of Texas Southwestern Medical Center microarray facility
Description
Control mice were given a 24-hour access for 30 days to two bottle of water
Data processing
We analyzed the array data using R programming language and the bioconductor packages (http://www.bioconductor.org). Preprocessing (RMA background correction and quantile normalization) of the microRNA data was performed with Bioconductor’s ‘Oligo’ package. Quality measures were taken before and after preprocessing using the Array Quality Metrics package (Kauffmann, Gentleman et al. 2009, Kauffmann and Huber 2010) to generate the principal component analysis. For all further analyses only the mouse mature microRNA data was used. The microarray expression data was then analyzed using the Bioconductor ‘Limma’ package (Smyth 2004). We used differential expression analysis between paired SN and TH samples (paired/dependent t-test) and between the alcohol and control samples in SN and TH (two independent t-tests). MicroRNA annotations, sequences, chromosomal locations and families were defined by miRbase version 17. We defined 10% as the threshold for determining if a certain chromosome was associated with a statistical measure and 5% as the threshold for determining if a microRNA family was associated.
