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-Ht-3
Label protocol
C: control diet 30: 30-month-old Ht: heart 3: sample number
Hybridization protocol
Total 10ug cRNA was placed in the gene chip. The gene chip I used for this study is the Affymetrix Murine Genome U74Av2 chips (Affymetrix, Santa Clara, CA), which covers 12,422 transcripts. 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
GeneChip Analysis Suite 5.0 was used to analyze the image data. The Affymetrix Mouse Genome U74Av2 chip is derived from selected genes and ESTs. Each gene is represented by the use of 16 perfectly matched and mismatched (MM) control probes. The MM probes acts as specificity controls that allows the direct subtraction of both background and cross-hybridization signals. To determine the quantitative RNA abundance, the average of the differences representing PM minus MM for each gene-specific probe family is calculated, after discarding the maximum, the minimum and any outliers beyond 3 standard deviations. To minimize variability in hybridization, the global scaling method normalizes signals of each chip. The global scaling method is computational technique in which the average signal of all probe sets in one chip is scaled to a target average intensity by multiplying a scaling factor. This scaling factor is multiplied to each probe set signal to give the raw expression.
