|
| Status |
Public on Aug 26, 2009 |
| Title |
liver_PQQ_5 |
| Sample type |
RNA |
| |
|
| Source name |
liver, PQQ supplemented
|
| Organism |
Rattus norvegicus |
| Characteristics |
strain: Sprague-Dawley
gender: male
tissue: liver
diet: PQQ supplemented
|
| Treatment protocol |
The effects of PQQ depletion and repletion were assessed using male rat pups derived from Sprague-Dawley dams fed the basal ASD (deficient or supplemented with PQQ). To produce PQQ deficiency, 10-week-old virgin Sprague-Dawley rats were mated and during the last 10 days of gestation, two-thirds of the rats were fed the basal ASD, while the remaining one-third were fed a PQQ supplemented diet (2 mg PQQ [6.67 nmol]/g diet). Offspring were then assigned to and fed the same diets as their corresponding dams. At 6 ½ weeks, half of the PQQ deficient (PQQ-) and supplemented rats (PQQ+) were next divided to generate short-term repleted (PQQ-/+) and depleted (PQQ+/-) groups. The repletion was accomplished by administering PQQ by i.p. injection at 1.5 mg PQQ/Kg body weight every 12 hours for a period of 36 hours. Short-term depletion was achieved by switching PQQ+ rats to the PQQ deficient diet for 48 hours. The depletion study was conducted for 48 hours to allow a washout period for cellular PQQ reserves. An additional group of rats (n=6) fed a standard laboratory chow diet were used as a reference.
|
| 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-labelled 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 |
PQQ supplemented rats.
1000_73553
|
| 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.
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| |
|
| Submission date |
Aug 25, 2009 |
| Last update date |
Aug 25, 2009 |
| Contact name |
eskouhi Tchaparian |
| E-mail(s) |
eskouhie@amgen.com
|
| Organization name |
UC-Davis
|
| Street address |
one shields avenue
|
| City |
davis |
| State/province |
CA |
| ZIP/Postal code |
95616 |
| Country |
USA |
| |
|
| Platform ID |
GPL2890 |
| Series (1) |
| GSE17811 |
Gene expression-based assessment of the effect of PQQ |
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