embryo: in vitro produced embryo - Day 6 of development
Treatment protocol
At day 5 after insemination, 5 µl of KSOM-BE2 or 5 µl of KSOM-BE2 containing 100 ng/ml CSF2 were added to each drop to achieve a final CSF2 concentration of 0 or 10 ng/ml. On Day 6, 24 hours after treatment, morulae and early blastocysts were selected and washed three times in 50 µl microdrops of 10 mM PO4 buffer, pH 7.4 containing 0.9% (w/v) NaCl and 1 mg/ml polyvinylpyrrolidone (PBS-PVP) by transferring the embryos from microdrop to microdrop. Embryos were frozen at -80°C in PBS-PVP in groups of 50. A total of 4 groups of embryos from each treatment were prepared in a total of 6 replicates of in vitro production.
Growth protocol
Cumulus oocyte complexes (COCs) from ovaries from a mixture of beef and dairy cattle were collected in Tissue Culture Medium-199 (TCM-199) with Hank’s salts without phenol red (Hyclone, Logan UT) supplemented with 2% (v/v) bovine steer serum containing 2 U/ml heparin, 100 U/ml penicillin-G, 0.1 mg/ml streptomycin, and 1 mM glutamine. Oocytes were allowed to mature for 20-22 h in groups of 10 in 50 µl microdrops of TCM-199 with Earle’s salts supplemented with 10% (v/v) bovine steer serum, 2 µg/ml estradiol ,17- 20 µg/ml bovine follicle stimulating hormone (Folltropin-V; Belleville, ON, Canada), 22 µg/ml sodium pyruvate, 50 µg/ml gentamicin sulfate, and 1 mM glutamine. Embryos were cultured at 38.5oC in a humidified atmosphere of 5% CO2 or 5% CO2, 5% O2, and 90% N2 (v/v). Cleavage rate was assessed at Day 3 after insemination.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted from embryos with the RNeasy Plus Micro kit (Qiagen-Inc, CA, USA) following manufacturer’s instructions. Concentration of the input RNA was determined by Nanodrop 1000 spectrophotometer (Thermo Scientific, Waltham, MA, USA) and RNA integrity was determined by Agilent 2100 Bioanalyzer with RNA 6000 Nano LabChip kit (Agilent Technologies, Santa Clara CA, USA). Only samples that showed RNA integrity > 7 were used for the microarray hybridization and quantitative PCR analysis. Extracted RNA was stored at -80oC until microarray analysis.
Label
Cy3
Label protocol
samples of RNA (1.5-3.0 pg/µl) were concentrated to 5 µl using Savant SpeedVac (Therno Scientific) at low heat and amplified into ss-cDNA using the NuGEN WT-Ovation Pico RNA amplification System (NuGen Technologies, Inc, San Carlos, CA, USA) following manufacturer's instructions. Amplified ss-cDNA was purified using Zymo Spin IIC columns (Zymo Research, Orange, California, USA) and stored at -20 oC overnight. Product yield and purity were determined by the Nanodrop 1000 spectrophotometer assuming that 1 absorbance unit at 260 nm of ss-cDNA is equal to 33 µg/ml. All 260/230 values were greater than 2. Aliquots containing 2 µg of ss-cDNA were labeled with the Agilent Genomic DNA Enzymatic Labeling kit to incorporate cyanine 3- or 5-labeled CTP. For half the replicates, ss-cDNA from control embryos was labeled with Cy3 and ss-cDNA from CSF2-treated embryos was labeled with Cy5. For the other replicates, control embryos were labeled with Cy 5 and CSF2-treated embryos with Cy3.
Hybridization protocol
Hybridizations were set up using 2 µg of each sample (Cy3 and Cy5 components) for a total of 4 µg per array. Volumes were brought to 44 µl with Diethylpyrocarbonate (DEPC)-water and then 11 µl of Agilent 10x GE Blocking Agent was added. The mixtures were incubated at 98oC for 3 minutes and then cooled to room temperature for 5 minutes before adding 55 µl of Agilent 2x Hi-RPM Hybridization buffer. Tubes were flash-spinned on a microfuge and lightly vortexed before loading 100 µl of the hybridization mixture onto each array. Hybridization was carried out for 17 h at 65°C and 10 rpm in a SureHyb gasket slide (Agilent). Washing and scanning procedures were carried out using standard Agilent guidelines for gene expression microarray processing.
Scan protocol
At the end of hybridization, microarray slides were sequentially washed using standard Agilent guidelines for gene expression microarray processing. Microarray slides were scanned immediately using an Agilent G2505B scanner.
Description
CFS2 treated embryos group 2
Data processing
Images were extracted and pre-processed using the Agilent Feature Extraction Software v 9.5 with default analysis parameters for the initial extraction, signal quantifications, and scaling of the generated data. The software produces background adjustment and normalizations for the dye of individual genes. The intensity of each spot was summarized as the median pixel intensity. All the generated values were then transformed to log2. The Lowess method was used for intensity normalization within each array. JMP® Genomics 3.1 for SAS® 9.1.3 software (SAS Inst., Inc., Cary, NC) was used for data global normalization and identification of differentially expressed genes. The PROC ANOVA procedure was used for simultaneous comparisons and the quantile method for intensity normalization. The model included replicate and treatment. Replicate (array) was considered random and treatment was considered fixed. Correction for false discovery rate was performed by the Benjamini and Hochberg method (1995). Only genes with median pixel intensity of at least 2.8 were considered to be expressed. Genes with a 1.5-fold difference and false discovery rate ≤ 0.01 were considered differentially expressed.
