strain: B6/C3H F1 hybrid gender: male age: 30-month-old
Biomaterial provider
University of Wisconsin-Madison, Genetics
Treatment protocol
control diet
Growth protocol
Male B6C3F1 mice were purchased from Harlan Sprague-Dawley at 6-7 weeks of age. Mice were housed singly in a pathogen-free facility and provided acidified water ad libitum.
Extracted molecule
polyA RNA
Extraction protocol
Total RNA was extracted from frozen tissue using the TRIZOL reagent (Life Technologies, Grand Island, NY) and mRNA was purified from the total RNA with oligo-dT-linked oligotex resin (Qiagen, Valencia, CA). Then, double-stranded cDNA was synthesized from 1ug of mRNA using the SuperScript Choice System (Life Technologies, Grand Island, NY) with an oligo-dT primer containing a T7 RNA polymerase promoter (Genset, La Jolla, CA), purified with phenol-chloroform-isoamyl alcohol, and precipitated with pellet paint co-precipitant (Novagen, Madison, WI). Purified double-stranded cDNA was used to synthesize biotin-labeled cRNA using the BioArray High Yield RNA Transcript Labeling Kit (Enzo, Farmingdale, NY). The biotin-labeled antisense cRNA was then purified with the RNeasy affinity column (Qiagen, Valencia, CA). After purification, biotin-labeled cRNA was fragmented randomly to sizes ranging from 35 to 200 bases.
Label
C30-Br-5
Label protocol
C: control diet 30: 30-month-old Br: brain(neocortex) 5: sample number
Hybridization protocol
Total 10ug cRNA was placed in the gene chip. The gene chip I used for this study is the Affymetrix MOE 430 2.0 arrays (Affymetrix, Santa Clara, CA). The Affymetrix MOE 430 2.0 array has 45,037 probe sets. Hybridization was carried out at 45ºC for 16 h with mixing on a rotisserie at 60 rpm. Following hybridization, the hybridization solutions were removed and the gene chips were be installed in a fluidics system for washes and staining.
Scan protocol
After staining, the gene chips were read at a resolution of 3 μm using a Hewlett Packard GeneArray Scanner (Affymetrix, Santa Clara, CA). The averaged images collected from two scanned images were used for the analysis.
Description
We used five animals per group, and hybridized each sample to independent DNA chips, because previous work from our laboratory suggests that variability between individuals is higher than variability observed in replicate hybridizations of the same samples.
Data processing
The GeneChip Operating Software (GCOS) version 1.2 was used to analyze the image data. GCOS determines the presence of mRNA in samples and computes the signals of probe sets using the differences and ratios between PM and MM signals. Signals in each image are normalized to minimize an overall variability in hybridization intensities by a global scaling method.
