gender: female tissue: Left ventricle cardiac tissue age: 12 months strain: generation 7 of HRT (see description for additional details)
Extracted molecule
total RNA
Extraction protocol
Left ventricle tissue was snap-frozen in liquid nitrogen and stored at -80oC. RNA was isolated from the left ventricle (20 mg) with the mirVana RNA isolation kit (Ambion) according to the manufacturer's instructions. RNA integrity, purity and quantity were assessed by Bioanalyzer (Agilent Technologies, Santa Clara, US) and Nanodrop (NanoDrop Technologies, Baltimore, US). The concentration of total RNA was measured by Nanodrop with ultraviolet spectrophotometry at 260/280 nm. RNA quality was assessed by electrophoresis on Bioanalyzer chips (Agilent Technologies, Santa Clara, US). Only samples with a 260/280-ratio of more than 1.8, and RNA integrity number (RIN) value above 8 was analyzed.
Label
Biotin
Label protocol
All samples were labeled at the same time and according to manufactures protocol
Hybridization protocol
Samples were hybridized to Applied Biosystems Rat Genome Survey chips v.1.0. The samples were hybridised over two days. The biological groups were balanced between the two hybridisation batches and the samples randomly distributed within this format. Samples were analyzed by the Applied Biosystems 1700 Array Expression System.
Scan protocol
Samples were analyzed by the Applied Biosystems 1700 Array Expression System.
Description
.The laboratory animals included in this study consists of rats (N:NIH outbred rat stock) selectively breed for low and high response to exercise based on their change in maximal treadmill running capacity, as estimated before and after 8 weeks of exercise training. This process has generated rat models with inherited differences for the adaptation component of aerobic capacity. The founder population, the N:NIH stock of rats, was developed in 1979 as a genetic resource of a heterogeneous population from the intentional outcross breeding of eight inbred strains, representing the broadest phylogenetic spectrum of laboratory rats available: Maudsely Reactor (MR/N), Wistar (WN/N), Wistar Kyoto (WKY/N), Marshall 520 (M520), Fischer (F344), Ax C 9935 Irish (ACI), Brown Norway (BN/SsN), and Buffalo (BUF/N). The resultant stock has wide genetic heterogeneity and is a superior choice as a starting population for artificial selection.
Data processing
The data files from the AB 1700 Chemiluminescent Microarray Analyzer Software were processed using J-Express Pro v.2.8. The signal intensity values were extracted per spot, and all flagged and control spots were filtered out. Before compiled into an expression profile data matrix, all arrays were quantile normalized to be comparable. Genes with at most 20% missing values were allowed in the final dataset. The signal intensities in the dataset were further log transformed, and missing values were replaced using the method LSimpute Adaptive. The search for differentially expressed genes was performed both on a single gene and gene set level. Rank Product was used to look for differentially expressed genes on a gene by gene basis, while Gene Set Enrichment Analysis (GSEA) was used to look for sets of genes sharing common characteristics that were differentially expressed between the classes examined.
