Cumulus-oocyte complexes (COC’s) were collected byaspiration with a 16G needle mount on an aspiration pump. The COC’s were denuded by vortexing, washed and stained with Hoechst 33342. Chromatin organization was evaluated under a inverted fluorescence microscope and classified according to the degree of chromatin condensation within the nuclear enveloppe. Oocytes were then washed, collected in small volumes of PBS, snap-frozen in liquid nitrogen and stored at -80°C until RNA extraction.
Growth protocol
Oocytes were collected from slaughterhouse-derived ovaries and grouped according to the chromatin configuration as described by Lodde et al. 2007.
Extracted molecule
total RNA
Extraction protocol
Total RNA extraction was performed using the PicoPure RNA isolation kit (Applied Biosystems, Carlsbad, CA, USA), with DNase treatment on the purification column according to the manufacturer instructions. RNA was eluted in 11 µl of elution buffer. Quantity and quality of each pool of RNA was verified on a 2100 Bioanalyzer (Agilent Technologies, Mississauga, On, Canada). Samples were stored at -80°C. RNA_amplication: Anti-sense RNA was produced using the RiboAmp HS RNA amplification kit (Applied Biosystems, Carlsbad, CA, USA). Quantity of aRNA was determined using a Nanodrop ND-1000 (NanoDrop Technologies, Wilmington, DE, USA).
Label
Cy5
Label protocol
Samples were labeled using the ULS Fluorescent Labeling Kit for Agilent arrays (with Cy3 and Cy5) (Kreatech Diagnostics, Amsterdam, The Netherlands). The labeled product was then purified using the picopure RNA extraction kit (Applied Biosystems, Carlsbad, CA, USA) to remove uncoupled dyes.
Cumulus-oocyte complexes (COC’s) were collected byaspiration with a 16G needle mount on an aspiration pump. The COC’s were denuded by vortexing, washed and stained with Hoechst 33342. Chromatin organization was evaluated under a inverted fluorescence microscope and classified according to the degree of chromatin condensation within the nuclear enveloppe. Oocytes were then washed, collected in small volumes of PBS, snap-frozen in liquid nitrogen and stored at -80°C until RNA extraction.
Growth protocol
Oocytes were collected from slaughterhouse-derived ovaries and grouped according to the chromatin configuration as described by Lodde et al. 2007.
Extracted molecule
total RNA
Extraction protocol
Total RNA extraction was performed using the PicoPure RNA isolation kit (Applied Biosystems, Carlsbad, CA, USA), with DNase treatment on the purification column according to the manufacturer instructions. RNA was eluted in 11 µl of elution buffer. Quantity and quality of each pool of RNA was verified on a 2100 Bioanalyzer (Agilent Technologies, Mississauga, On, Canada). Samples were stored at -80°C. RNA_amplication: Anti-sense RNA was produced using the RiboAmp HS RNA amplification kit (Applied Biosystems, Carlsbad, CA, USA). Quantity of aRNA was determined using a Nanodrop ND-1000 (NanoDrop Technologies, Wilmington, DE, USA).
Label
Cy3
Label protocol
Samples were labeled using the ULS Fluorescent Labeling Kit for Agilent arrays (with Cy3 and Cy5) (Kreatech Diagnostics, Amsterdam, The Netherlands). The labeled product was then purified using the picopure RNA extraction kit (Applied Biosystems, Carlsbad, CA, USA) to remove uncoupled dyes.
Hybridization protocol
825 ng of each labeled sample were incubated in a solution containing 2x blocking agent and 5x fragmentation buffer in a volume of 55 µl at 65°C for 15 minutes and were then put on ice. 55 µl of 2x GEx Hybridization Buffer HI-RPM was added for a final volume of 110 µl. The hybridization mix (100 µl) was added onto the array and hybridization was performed at 65°C for 17 h using an Agilent Hybridization chamber in a rotating oven. After the hybridization step, slides were washed with gene expression wash buffer 1 containing 0.005% triton X-102 for 3 minutes at room temperature and then transferred to gene expression wash buffer 2 containing 0.005% triton X-102 for 3 minutes at 42°C. Final washes with acetonitrile for 10 sec at room temperature and with drying and stabilisation solution for 30 sec at room temperature were performed before air-drying the slides.
Scan protocol
Scanned on an Tecan Power scanner (Tecan group ltd.)
Data processing
Images were quantified using ArrayPro 6.4 software (Media Cybernetics). Background subtracted, LOESS normalized, data obtained from log2 of processed Red signal/processed Green signal.
