Regassa et al. BMC Genomics 2011, 12:57 http://www.biomedcentral.com/1471-2164/12/57 biological replicates; each replicate containing 50 oocytes or CCs samples (Figure 1). In experiment 1a, CCs were mechanically removed from oocytes at GV stage by repeated in and out pipetting and the resulting denuded oocytes (DOs), designated by number 1 and their companion CCs by number 2, were frozen. In experiment 1b, intact COCs were cultured; their compa- nion CCs were mechanically removed and the resulting denuded MII oocytes, designated by number 4 and their companion CCs by number 3, were frozen. In experi- ment 2, CCs were mechanically removed from their enclosed oocytes at GV stage and the resulting (OO - CCs), designated by number 5 and other intact COCs (OO + CCs) were cultured separately. After 22 hr cul- ture period, CCs were mechanically removed from the intact oocytes and the resulting denuded MII oocytes (number 6) were frozen. In experiment 3, oocytes were micro surgically removed from their companion CCs at GV stage as described previously [17,46,47]. Briefly, using micromanipulators with a holding pipette (20 pm diameter hole) and a fine glass needle, the complex is held under negative pressure and the oocyte pierced through both sides of the zona pellucida. The contents of the oocyte are then aspirated through the holding pipette while removing the glass needle, resulting in temporary deformation of the zona pellucida. Almost all of the oocyte contents are removed in this way, leaving an intact complex without the oocyte, the oocytecto- mised complex (CCs - OO). The resulting complexes (CCs - OO), designated by number 7 and other intact complexes (CCs + OO) were cultured. After 22 hr cul- ture, oocytes were micro surgically removed from their intact cumulus oocyte complexes (CCs + OO), as Figure 1 Diagrammatic illustration of the experimental design showing groups that were used for total RNA isolation, cDNA synthesis, array hybridization and quantitative and semi- quantitative RT-PCR validation of the array data. Numbers, 1 - 10, under each oocyte and CCs figure, represent the samples that were used for RNA isolation, cDNA synthesis and array hybridisation, as explained in materials and methods section. For all experimental groups, the samples were derived from GV stage COCs. Page 3 of 20 described above and the resulting MII CCs without their enclosed oocytes, designated by number 8 were frozen. In experiment 4, CCs were mechanically removed from their enclosed oocytes both at GV (num- ber 9) and MII stages (number 10) and frozen for subse- quent total RNA isolation. In both cases, cells were cultured for 22 hrs and each experiment was repeated three times. Complete removal of either cell from one or the other was confirmed by microscopic examination of the corresponding samples (Figures 2A and 2B) as described in [53]. Cells were cultured in groups of 50 in 400 μl MPM medium supplemented with 12% estrus cow serum and 10 μg mL-1 FSH for 22 hr at 39°C in an incubator with humidified atmosphere containing 5% CO2. Samples were stored at -80°C until subsequent RNA isolation. RNA isolation and array processing In all experiments, always triplicate pools of oocytes or their corresponding CCs (each pool contains 50 oocytes or their corresponding CCs), were used for RNA isola- tion and subsequent downstream analysis. The total oocytes or CCs samples used for this study correspond to 150 oocytes or their respective CCs per each experiment. Total RNA was isolated using PicoPure RNA Isolation Kit according to the manufacturer’s instruction (Arc- turus Bioscience Mt. View CA). Briefly, the samples were extracted using 100 μL of extraction buffer and resuspended by pipetting gently. They were incubated for 30 min at 42°C and centrifuged at 3,000 × g for two min. One hundred μL of 70% ethanol was pipetted to the cell extract and mixed well by pipetting up and down. Two hundred μL conditioning buffer was pipetted onto the purification column filter membrane and the columns were incubated with conditioning buffer for 5 min at room temperature. The purification columns were centrifuged in the provided collection tube at 16,000 × g for 1 min and 50 μL of 70% ethanol was pipetted into the cell extract. The cell extract and Figure 2 Representative images showing CCs from which the enclosed oocyte was completely removed at GV stage using oocytectomy (A) and the oocyte from which the companion CCs were completely removed mechanically at GV stage using pipettes (B) and intact COC (C). Images were taken using Leica DM-IRB microscope with a magnification of 100×.