Strain: Inbred Short Sleep Gender: Male Tissue: Cerebellum Alcohol Exposure: Naive
Biomaterial provider
Mouse from the Institute of Behavioral Genetics, Boulder, CO
Treatment protocol
Food and water ad libitum for 14 days after transfer from IBG to UCHSC.
Extracted molecule
total RNA
Extraction protocol
Qiagen RNeasy Total RNA Isolation Kit
Label
Biotin
Label protocol
Total RNA (10 µg) is converted to double-stranded cDNA (ds-cDNA) by using the Superscript Choice System (Life Technologies Inc., Gaithersburg, MD, USA). An oligo-dT primer containing a T7 RNA polymerase promoter (Proligo Primers & Probes, Boulder, Colorado, USA) is utilized. The ds-cDNA is purified and recovered by using GeneChip sample cleanup module (Affymetrix).In vitro transcription is performed to generate biotin-labeled cRNA. In vitro transcription is performed using a RNA Transcript Labeling Kit (Enzo, Farmingdale, New York, USA) and ds-cDNA template is transcribed in the presence of a mixture of biotin-labeled ribonucleotides. Biotin-labeled cRNA is purified using GeneChip sample cleanup module (Affymetrix) or an RNeasy affinity column (Qiagen). To ensure optimal hybridization to the oligonucleotide array, the cRNA is fragmented. Fragmentation is performed such that the cRNA fragments are between 50-200 bases in length by incubating the cRNA at 94 °C for 35 min in a fragmentation buffer. The sample is then added to a hybridization solution containing 100 mM MES, 1 M Na+, and 20 mM EDTA in the presence of 0.01% Tween 20. The final concentration of the fragmented cRNA is 0.05 µg/µL.
Hybridization protocol
Hybridization is performed by incubating 200 ?L of the sample to the Affymetrix GeneChip® arrays (Affymetrix Inc., Santa Clara, California, USA). Hybridization occurs at 45 °C for 16 hours using a GeneChip® Hybridization Oven 640 (Affymetrix). After hybridization, the hybridization solutions are removed and the arrays are washed and stained with Streptavidin-phycoerythrin using a GeneChip® Fluidics Station 450 (Affymetrix).
Scan protocol
Arrays are read at a resolution of 2.5 to 3 microns using the GeneChip Scanner 3000 (Affymetrix).
Description
Assay of baseline expression in the cerebellum of an alcohol-naive Inbred Short Sleep mouse
