Subsequent to storage, samples were thawed and washed twice in 2 ml of PBS, then suspended in round cell lysis buffer (0.1% SDS, 0.5% Triton X-100 in RNase free H2O). RNA was extracted using the RNeasy mini kit (Qiagen GmbH., Germany) with gentle modifications. Sperm cells were suspended in 600 µl of lysis buffer for 107 cells. This cell suspention was lysed using a Politron as previously described (Saade, et al., 2007), to maximize the recovery of spermatozoal RNA. The lysates were then processed according to the manufacturer’s instructions using the animal cell protocol. After RNase-free DNase treatment (Qiagen GmbH., Germany), the RNA was eluted with 30µl. RNA concentration was determined using a nanodrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). RNA integrity was assessed on an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA).
Label
Cy3
Label protocol
Cyanine-3 (Cy3) labeled cRNA was prepared from 0.5 ug RNA using the One-Color Low RNA Input Linear Amplification PLUS kit (Agilent) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
Hybridization protocol
1.5 ug of Cy3-labelled cRNA (specific activity >10.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 250 ml containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 250 ml of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Whole Human Genome Oligo Microarrays (G4112A) for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent), then dried immediately by brief centrifugation.
Scan protocol
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505B) using one color scan setting for 1x44k array slides (Scan Area 61x21.6 mm, Scan resolution 10um, Dye channel is set to Green and Green PMT is set to 100%).
Description
dataNorm_251485046835_1_3 Tobacco-induced gene expression alterations in human spermatozoa
Data processing
The scanned images were analyzed with Feature Extraction Software 9.1 (Agilent) using default parameters (protocol GE1-v1_91 and Grid: 012391_D_20060331) to obtain background subtracted and spatially detrended Processed Signal intensities. All data were normalized by quantile normalization.
Tobacco-induced microRNAs and mRNAs profile alterations in human spermatozoa: a preliminary study for further knowledge of toxical spermatogenesis impairment
