Whole testes were obtained from animals at different ages depending on the experiment. Immediately after removal, testis samples were snap-frozen and stored in liquid nitrogen. Before RNA extraction, testis samples were weighed and homogenized in a Dounce homogenizer (Kontes Co., Vineland, NJ). Thereafter, RNA was isolated with the RNeasy Kit (Qiagen, Chatsworth, CA) according to the manufacturer’s instructions, encompassing an on-column deoxyribonuclease I (DNase I) treatment of the RNA. For quantitative RT-PCR analyses, 5ng luciferase mRNA (Promega Corp., Madison, WI) were added to each testis sample before RNA preparation as an external standard. The quality of all testis RNA samples was monitored by measuring the 260/280 and 260/230 nm ratios with a Nano-Drop spectrophotometer (NanoDrop Technologies, Centreville, DE) and by means of the Agilent 2100 BioAnalyzer (Agilent, Palo Alto, CA). Only RNA showing no signs of degradation or impurities (260/280 and 260/230 nm ratios, larger than 1.8) was considered suitable for microarray analysis.
Label protocol
Before target synthesis, RNA extracts from one testis of three control or three SCARKO littermates were pooled, creating paired control and SCARKO samples optimally adjusted for potential variation between litters. Five micrograms of polyadenylated RNA from each pool was reverse transcribed into double-stranded DNA with a polydeoxythymidine-T7 primer using SuperScript II RT, ribonuclease H, and DNA polymerase I (Invitrogen Life Technologies, Inc., Carlsbad, CA). From this double-stranded DNA, biotin-labeled target aRNA was generated in a T7 in vitro transcription reaction using the Affymetrix IVT Labeling Kit (Affymetrix, High Wycombe, UK).
Hybridization protocol
After purification (GeneChip Sample Cleanup Module, Affymetrix), yield (30–120 µg) and purity (260/280 and 260/230 nm ratios, >1.8) of the labeled amplified RNA were analyzed, 20 µg quality-controlled amplified RNA was fragmented by alkaline hydrolysis and resuspended with control spikes in 300 µl hybridization buffer (Eukaryotic Hybridization Control Kit, Affymetrix). Of this probe solution, 200 µl was used for hybridization in a rotisserie oven at 45 C.
Scan protocol
After hybridization, gene chips were washed and stained in the GeneChip Fluidics Workstation 400 (Affymetrix) using the EukGE-WS2v4 protocol and subsequently scanned with the GeneChip Scanner 3000 (Affymetrix). Image analysis was performed by use of Affymetrix GeneChip operating software.
Description
RNA from 1 testis from three 10 day old control (AMHCre) mice. Immediately after removal, testis samples were snap-frozen and stored in liquid nitrogen. Before RNA extraction, testis samples were weighed and homogenized in a Dounce homogenizer. Thereafter RNA was isolated with the QIAGEN RNeasy Kit (Chatsworth, CA) according to the manufacturers instructions, encompassing an on-column DNaseI-treatment of the RNA. Keywords = SCARKO Keywords = testis Keywords = spermatogenesis Keywords = androgens Keywords = microarray Keywords = Pem Keywords = serine protease inhibitor
Data processing
Expression values were computed with the Affymetrix Microarray Suite (MAS) 5.0 software. The amount of transcripts in a sample that scored present, absent, or marginally detectable was assessed, also with the MAS 5.0 software.
