source1 = Pooled RNA samples correspond to 4 males and 4 females after 20 weeks of endurance exercise-training, with the following characteristics (mean±sd): Age 29.2 ±14.7 yrs, BMI 24.6 ±5.0 kg/m2, insulin sensitivity 1.7±0.3 μU/ml/min. These individuals had no changes or lower their insulin sensitivity response (LSIR) after 20 wk of endurance exercise-training, the average response was –0.8±1.7 μU/ml/min. Cy5 labeled.
source2 = Pooled RNA samples correspond to 3 males and 4 females after 20 weeks of endurance exercise-training, with the following characteristics (mean±sd): age 29.2 ±12.9 yrs, BMI 23.2 ±2.5 kg/m2, insulin sensitivity 2.9±0.5 μU/ml/min. These individuals had high insulin sensitivity response (HSIR) after 20 wk of endurance exercise-training, their average response was +4.4±0.5 μU/ml/min. Cy3 labeled.
Muscle biopsies were taken from the middle of the vastus lateralis by the percutaneous needle biopsy technique. Frozen tissue samples were crushed in liquid nitrogen, and total RNA was extracted using the TRI Reagent followed by purification in Qiagen columns from the RNeasy kit. Equal amounts from the individual RNA samples were used to generate RNA pools for this experiments using the HSIR (1) and LSIR (2) sources. Pool RNA samples (2 µg, each pool) was reverse-transcribed into cDNA with reagents provided by the MICROMAX Human cDNA Microarray System. These RNA pools were used for the biotin or fluorescein labeling to generate the cDNAs analyzed in the microarrays. The differentially fluorescent-labeled first-strand cDNA from each source was suspended in 22 µl of hybridization solution containing 5µl of poly-A (5mg/ml), 12.5µl of 20x SSC, 0.5µl 10% SDS and 12.5µl deionized formamide, combined and hybridized to the arrays for 30-36 hrs. The signals from specifically hybridized biotin- and fluorescein-labeled cDNAs were amplified either with streptavidin–horseradish peroxidase (HRP) and Cy5–tyramide or antifluorescein–HRP and Cy3–tyramide, respectively. Fluorescent array images were collected with a GSI Lumonics ScanArray 5000 fluorescent scanner (Packard Biochip Technologies; Meriden, CT) and image intensity data were extracted and analyzed using the provided QuantArray V. 3.0 software (Packard Bioscience; Meriden, CT). A uniform scale factor was applied to normalize signal intensities between Cy5 and Cy3. LOWESS normalization was applied to account for dye variation across and between slides, and for signal intensity effects. An in-house software program was used to perform this analysis (Details of the algorithm can be found at http://bioinfo.pbrc.edu). Fluorescent intensities below 500 were removed, and the expression ratios were lowess transformed to obtain the log base 2 values given in the data table. Keywords = skeletal muscle Keywords = vastus lateralis Keywords = healthy individuals Lot batch = PBRC series H3-47
