gender: male strain: C57Bl/6J treatment: ethanol tissue: hippocampus
Treatment protocol
All protocols were approved by the Animal Use Subcommittee (AUS) at the University of Western Ontario, London, Ontario, Canada. C57BL/6J (B6) mice were obtained from Jackson Laboratories (Bar Harbor, MA) and a population was subsequently maintained at the Animal Care Facility at the University of Western Ontario. Female mice 12-18 weeks of age were separated into individual cages and mated with males of approximately the same age. The day of birth was termed post-natal day (PD) zero. Sex and weight-matched littermate pups were divided into two groups: ethanol-treated and saline control mice. Pups were given two subcutaneous dorsal injections at 9 am and 11 am on both PD4 and PD7. Ethanol-treated mice were injected with 2.5 g/kg of ethanol in 0.15 M NaCl. Pups were weaned on PD21 and housed in cages of two to four same-sex littermates. Male mice were used for all subsequent analyses (n=18). Mice were sacrificed on PD 70 via carbon dioxide asphyxiation. The hippocampus was dissected out, snap-frozen in liquid nitrogen, and stored at -80°C
Extracted molecule
total RNA
Extraction protocol
Qiagen Allprep DNA RNA kit was used according to Qiagen protocol
Label
Biotin
Label protocol
All sample labeling and GeneChip processing was performed at the London Regional Genomics Centre (Robarts Research Institute, London, Ontario, Canada; http://www.lrgc.ca). RNA quality was assessed using the Agilent 2100 Bioanalyzer (Agilent Technologies Inc., Palo Alto, CA) and the RNA 6000 Nano kit (Caliper Life Sciences, Mountain View, CA). Single stranded complimentary DNA (sscDNA) was prepared from 200 ng of total RNA as per the Ambion WT Expression Kit for Affymetrix GeneChip Whole Transcript WT Expression Arrays (http://www.ambion.com/techlib/prot/fm_4411973.pdf, Applied Biosystems, Carlsbad, CA) and the Affymetrix GeneChip WT Terminal Labeling kit and Hybridization User Manual (http://media.affymetrix.com/support/downloads/manuals/wt_term_label_ambion_user_manual.pdf, Affymetrix, Santa Clara, CA). Total RNA was first converted to cDNA, followed by in vitro transcription to make cRNA. 5.5 ug of single stranded cDNA was synthesized, end labeled
All washing steps were performed by a GeneChip Fluidics Station 450 and GeneChips were scanned with the GeneChip Scanner 3000 7G (Affymetrix, Santa Clara, CA) using Command Console v1.1.
Description
miRNA expression data
Data processing
Probe level (.CEL file) data was generated using Affymetrix Command Console v1.1. Probes were summarized to the miRNA and gene level using RMA
