tissue: Whole blood gender: Male age: 52 subject: athlete time point: before exercise
Treatment protocol
Whole blood samples for each participant were stored in PAXgene blood RNA tubes (Qiagen GmbH, Germany) at -80 °C from all three time-points until further analysis.
Extracted molecule
total RNA
Extraction protocol
RNA was isolated from blood sample aliquots (2.5 ml) using a PAXgene blood RNA kit (Qiagen GmbH, Germany) according to the manufacturer’s protocol. Residual DNA was removed with DNase I treatment, according to the manufacturer’s instructions (Qiagen GmbH, Hombrechtikon, Germany). The quality of the isolated RNA was checked with Agilent RNA 6000 Nano Assay kit reagents using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Samples with a RNA Integrity Number (RIN) of 7.0 or higher were used for further analysis. The RNA samples were quantified using a standard Nanodrop ND-1000 spectrophotometer (Nanodrop, Wilmington, DE, USA).
Label
Cy3
Label protocol
Total RNA (200 ng) from each sample was spiked with One-Color RNA Spike-In Kit reagents (Agilent Technologies, Santa Clara, CA, USA) according to the manufacturer’s instructions.Subsequent amplification and labeling of the samples to generate fluorescent cRNA (complimentary RNA) were carried out using the One-Color Quick Amp Labeling Kit (Agilent Technologies, Santa Clara, CA, USA) according to the manufacturer’s instructions.Labeled cRNA was purified using an RNeasy Mini kit (Qiagen). Only cRNA samples with a yield >1.65 µg, and a specific activity >9 pmol Cy3/µg cRNA were used for subsequent hybridization onto microarray slides.
Hybridization protocol
Labeled RNA was hybridized to Agilent whole genome expression array (4x44K) at 65°C for 17h in rotisserie in a hybridization oven. Disassembled arrays were washed in GE wash buffer 1&2 for 1 minutes each.
Scan protocol
Images were scanned with Agilent scanner at 5µm resolution.
Description
Gene expression in blood of Athlete 2 before exercise, replicate 2
Data processing
Raw data obtained from the microarrays were imported into Nexus Expression 3.0 (BioDiscovery Inc, Hawthorne, CA, USA) and processed according to the default settings of the software, which included a local background correction, removal of flagged spots and performing quantile normalization.
