Rats were fed an amino acid semi-purified based diet (ASD), which provided all known required nutrients in sufficient quantities to provide maximal growth, reproduction, and lactation. The ASD contained < 5 fmol PQQ/g. To assess the response to PQQ, male Sprague-Dawley rats, (Charles River Laboratory, Inc., Wilmington, MA) weighing ~250 grams (7-8 weeks old) were fed the ASD for two weeks. Groups of rats (n = 6/group) were then challenged for an additional two weeks with PQQ added at 2.5 mg PQQ/Kg ASD. For comparisons, separate groups (6 rats each) were fed epicatechin (Epi) added at 1000 mg Epi/kg ASD, or injected i.p. with dexamethasone (Dex, 100 mg Dex/Kg body wt), clofibrate (Clo, 100 mg Clo/Kg body wt), or phenobarbital (Pb, 80 mg Pb/Kg body wt) 3 hours prior to harvesting liver tissue .
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated from the liver of each rat using Trizol reagent (Invitrogen, Carlsbad, CA) and further purified using Qiagen RNA mini-kits (Qiagen, Valencia, CA). On-column DNA digestion using RNAse Free DNAase kit (Qiagen, Valencia, CA) was performed to remove DNA residues.
Label
Biotin
Label protocol
Labeling was performed using standard procedures and instructions for the Codelink® Array Bioassays. The biotin-labeled cRNA target was prepared by a linear amplification method. The poly(A)+ RNA subpopulation (within the total RNA population) was primed for reverse transcription by a DNA oligonucleotide containing the T7 RNA polymerase promoter 5′ to a d(T)24 sequence. After second-strand cDNA synthesis, the cDNA serves as the template for an in vitro transcription (IVT) reaction to produce the target cRNA. The IVT was performed in the presence of biotinylated nucleotides to label the target cRNA.
Hybridization protocol
Hybridization was performed using the CodeLink Expression Assay Reagent Kit and following the standard procedure for the Codelink Array Bioassays.
Scan protocol
Scanning and image analyses were performed with Axon’s 4000B using CodeLink’s Expression Analysis software.
Description
Pooled liver RNA from 6 rats injected i.p. with dexamethasone (Dex, 100 mg Dex/Kg body wt) 3 hours prior to harvesting liver tissue
Data processing
Data exported from the CodeLink analysis software was normalized using global Loess normalization where signal intensities from each array are Loess normalized to the average intensity of all arrays after removing high variance data points. Data from the individual single-dye experimental and reference arrays were combined and intensity values averaged and compared within and between groups to generate gene expression ratios along with t-test p-values and intensity-based ratio Z-scores. Clustering of the data was performed with a partitioning algorithm substantially similar to the CAST algorithm (34), but with a dynamically chosen threshold set to the 15th percentile value of 500 randomly chosen gene expression distances. These gene expression distances were calculated as cosine angle distances between the gene expression fold change vectors.
