tissue: Caput distal segment donor number: 1 donor age: 37
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated from each segment of caput, corpus and the cauda epididymal regions of human epidydimis from 3 healthy patients. Frozen tissues were powdered with a pestle and mortar on dry ice, and homogenized in RLT lysis buffer (Qiagen). RNA was purified with the RNeasy Mini Kit (Qiagen) according to the manufacturer's protocol, and potential genomic DNA contamination was eliminated with the RNase-free DNase set (Qiagen). Total RNA was eluted in RNAse-free water and its concentration was quantified with a NanoDrop 1000 microvolume spectrophotometer (Thermo Scientific). RNA quality was assessed with the Agilent RNA 6000 pico and Agilent RNA nano kits on a 2100 Bioanalyzer from the transcriptomic Core facility of the CHUQ (Agilent). Samples were stored at −80°C until use.
Label
Biotin
Label protocol
For each sample, 400 ng of total RNA were labeled with the FlashTag Biotin HSR Labeling Kit (Affimetrix, Santa Clara, USA). Microarray analyses were carried out using an Affymetrix GeneChip Human Clariom S array (Affymetrix, CA, USA).
Hybridization protocol
Fifteen micrograms of fragmented cRNA were hybridized for 16 hrs at 45°C under constant rotation. After hybridization, chips were processed using the Affymetrix GeneChip Fluidic Station 450 (protocol EukGE-WS2v5_450). Staining was made with streptavidin-conjugated phycoerythrin (SAPE, Molecular Probes), followed by amplification with a biotinylated anti17 streptavidin antibody (Vector Laboratories), and followed by a second round of SAPE.
Scan protocol
The arrays were scanned using the Affymetrix GCS 3000 7G and the Gene-Chip Operating Software (Affymetrix, Santa Clara, CA), to produce the intensity files. Microarray hybridization was carried out at the Microarrays Facility of the Research Center of Laval University CRCHUL.
Data processing
Background subtraction and normalization of probe set intensities were performed using the method of Robust Multiarray Analysis (RMA) described by Irizarry et al. (Irizarry et al, 2003).
