Sperm isolation protocol: The cauda epididymides were punctured repeatedly with 30 or 26 gauge needles and put into micro-centrifuge tubes containing 1 mL phosphate-buffered saline (PBS). The samples were incubated in a water bath at 37°C for 10 minutes to aid sperm diffusion from the tissue. The tubes were spun for 3 minutes at 300 xg to pellet the epididymal pieces and the supernatant containing sperm were put into a new tube. The tube was spun for an additional 5 minutes at 2000 xg to pellet the sperm and the supernatant was removed. One mL of lysis buffer (0.15 M ammonium chloride, 10 mM potassium bicarbonate, 0.1 mM EDTA) was added to the sperm pellet, incubated on the bench top for 30 seconds and spun at 16,100 xg for 1 minute. The pellet was washed with 1 mL of PBS and spun at 16,100 xg for 1 minute.
RNA was extracted from the fresh sperm using the mirVana miRNA Isolation Kit (Applied Biosystems/Ambion, Austin, TX, USA), which separated the RNA into large and small fractions, per manufacturer’s instructions.
Label
biotin
Label protocol
Followed manufacturer's instructions.
Hybridization protocol
Followed manufacturer's instructions.
Scan protocol
Followed manufacturer's instructions.
Description
messenger RNA
Data processing
The probe cell intensity data from the Affymetrix GeneChips was normalized and annotated following previously published methods (Pacheco, Houseman et al. 2011 (PMID 21674046)). The linear models of microarray analysis (LIMMA) procedure (Smyth, Yang et al. 2003) (R package limma) was used to fit a linear regression model for each transcript on the Affymetrix GeneChip Array for each pair of treatment and controls. The p-values were adjusted for multiple comparisons with the qvalue package in R (Storey 2002) and the log2 expression ratios were transformed into fold change values.
