Ovaries from adult animals were collected at local abattoirs.
Growth protocol
The cumulus-oocyte-complexes (COCs) were aspirated from all visible 3-7 mm follicles, selected (those with more than four compact layers of cumulus cells and homogenous cytoplasm), washed three to four times in Hepes-buffered hamster embryo culture (HH) medium and cumulus cells were completely separated by hyaluronidase (0.1%) digestion and repeated pipetting. The separated cumulus cells (four pools of cumulus cells from 18-20 oocytes each from adult and prepubertal animals) were snap frozen in 100 µl of lysis solution (RNAqueous Micro Kit, Ambion Inc., Austin, TX) and stored at -80 C until RNA isolation and subsequent microarray analysis.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted from each of the cumulus cell samples using the RNAqueous micro kit (Ambion) according to manufacturer’s instructions. RNA was eluted twice using 10 µl volume of prewarmed (75 C) elution solution according to manufacturer’s instructions. Residual genomic DNA in all extracted samples was removed by DNAse I digestion (Ambion). The RNA from each pool of cumulus samples was divided into two 10 µl aliquots. One aliquot of extracted total RNA was used for cDNA microarray analysis. Total RNA (10 µl) from pools of cumulus cells (n = 4) was amplified using the RiboAmp Kit (Arcturus, Mountain View, CA) using previously validated procedures [Patel et al., Vet Immunol Immunopathol 2005; 105: 331-342]. The quality and quantity of the amplified RNA generated were estimated using a u.v. spectrophotometer (Beckman Instruments, Fullerton, CA) and the Bioanalyzer 2100 RNA 6000 nanochip (Agilent Technologies, Walbronn, Germany). Only those amplification reactions yielding amplified RNA of consistent size range and quality across samples were utilized in subsequent microarray experiments.
Label
Cy3
Label protocol
A total of 15 µg amplified RNA from cumulus samples were used for cDNA synthesis and labeling [Patel et al., Vet Immunol Immunopathol 2005; 105: 331-342]. Amplified RNAs were used for cDNA synthesis and labeling using the Clontech PowerScript Fluorescent Labeling System (BD Biosciences, Alameda, CA, USA) with oligo (dT)18 as primer. Synthesis of cDNA in this system incorporates an amino-modified dUTP into the cDNA. Following first strand synthesis, cDNAs were labeled using n-hydroxysuccinate (NHS-) derivitized Cy3 and Cy5 dyes (Amersham Biosciences Corp., Little Chalfont, England). Labeled cDNAs were purified to remove unincorporated dyes, combined and concentrated to approximately 10 μl using Microcon 30 spin concentrators (Millipore Corp., Bedford, MA, USA).
Ovaries from prepubertal animals were collected at local abattoirs.
Growth protocol
The cumulus-oocyte-complexes (COCs) were aspirated from all visible 3-7 mm follicles, selected (those with more than four compact layers of cumulus cells and homogenous cytoplasm), washed three to four times in Hepes-buffered hamster embryo culture (HH) medium and cumulus cells were completely separated by hyaluronidase (0.1%) digestion and repeated pipetting. The separated cumulus cells (four pools of cumulus cells from 18-20 oocytes each from adult and prepubertal animals) were snap frozen in 100 µl of lysis solution (RNAqueous Micro Kit, Ambion Inc., Austin, TX) and stored at -80 C until RNA isolation and subsequent microarray analysis.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted from each of the cumulus cell samples using the RNAqueous micro kit (Ambion) according to manufacturer’s instructions. RNA was eluted twice using 10 µl volume of prewarmed (75 C) elution solution according to manufacturer’s instructions. Residual genomic DNA in all extracted samples was removed by DNAse I digestion (Ambion). The RNA from each pool of cumulus samples was divided into two 10 µl aliquots. One aliquot of extracted total RNA was used for cDNA microarray analysis. Total RNA (10 µl) from pools of cumulus cells (n = 4) was amplified using the RiboAmp Kit (Arcturus, Mountain View, CA) using previously validated procedures [Patel et al., Vet Immunol Immunopathol 2005; 105: 331-342]. The quality and quantity of the amplified RNA generated were estimated using a u.v. spectrophotometer (Beckman Instruments, Fullerton, CA) and the Bioanalyzer 2100 RNA 6000 nanochip (Agilent Technologies, Walbronn, Germany). Only those amplification reactions yielding amplified RNA of consistent size range and quality across samples were utilized in subsequent microarray experiments.
Label
Cy5
Label protocol
A total of 15 µg amplified RNA from cumulus samples were used for cDNA synthesis and labeling [Patel et al., Vet Immunol Immunopathol 2005; 105: 331-342]. Amplified RNAs were used for cDNA synthesis and labeling using the Clontech PowerScript Fluorescent Labeling System (BD Biosciences, Alameda, CA, USA) with oligo (dT)18 as primer. Synthesis of cDNA in this system incorporates an amino-modified dUTP into the cDNA. Following first strand synthesis, cDNAs were labeled using n-hydroxysuccinate (NHS-) derivitized Cy3 and Cy5 dyes (Amersham Biosciences Corp., Little Chalfont, England). Labeled cDNAs were purified to remove unincorporated dyes, combined and concentrated to approximately 10 μl using Microcon 30 spin concentrators (Millipore Corp., Bedford, MA, USA).
Hybridization protocol
100 ul of pre-warmed (70°C) hybridization buffer (Slidehybe 3, Ambion) was added to probe mixtures and final probe solutions applied to pre-warmed microarray slides. Hybridizations were conducted for 18 hours using step down temperatures from 65°C to 42°C in sealed chambers of a Gene Tac hybridization chamber (Genomic Solutions, Inc., Ann Arbor, MI, USA). Following hybridization, slides were washed 2X with medium stringency buffer, 1X with high stringency buffer (Genomic Solutions), rinsed briefly at room temperature in 2X saline sodium citrate and then water, and dried by centrifugation at 1000 RPM in a cushioned 50-ml conical centrifuge tube.
Scan protocol
Dried microarrays were scanned immediately using a GeneTac LS IV microarray scanner (Genomic Solutions). GeneTac Integrator 4.0 software was then used to process array images, align spots, integrate robot-spotting files with the microarray image, and to export reports of spot intensity data.
Description
Bovine adult versus prepubertal cumulus cells, Replicate 4
Data processing
For microarray experiments, estimates of FDR and differentially expressed genes (FDR = 5%) were identified using the significance analysis of microarrays program [34]. Differences in ratios of Cy3/Cy5 < 0.7 (P < 0.05; FDR = 5%) or Cy3/Cy5>1.3 (P < 0.05; FDR = 5%) were considered significant. Initial annotation and gene symbols for features on the array were obtained using the Gene Links toolkit (http://cafg.msu.edu) and Unigene search function (http://www.ncbi.nlm.nih.gov/sites/entrez?db=unigene). Identification of biological themes (overrepresented genes) within lists of genes showing greater mRNA abundance in cumulus cells of prepubertal and adult oocytes was performed using expression analysis systematic explorer (EASE) [35] with a FDR of 10%.
