source: in-vitro culture media that did not contact any embryos (negative control)
Growth protocol
Bovine embryos were in-vitro produced following standard laboratory procedures. Briefly, ovaries were obtained from the local abbotoir and oocytes were retrieved and matured for 24 hours prior to co-culture with bovine sperm for in-vitro fertilization. After 18 hours, presumptive zygotes were transferred to in-vitro culture media and grown in groups of 20 to the desired time stage of development. 2-cell, 8-cell, and blastocyst embryos were cultured for 18-30 hours, 60-80 hours, and 168-192 hours post fertilization. At each time point, embryos were remove from the in-vitro culture media and approximetely 25 µL of spent in-vitro culture media was extracted from each in-vitro culture drops. Spent media samples from each developmental stage was pooled together, flash frozen in liquid nitrogen, and stored at -80 ˚C until total RNA extraction.
Extracted molecule
total RNA
Extraction protocol
Qiazol extraction for total RNA extract was performed as per manufacturer's instructions
Label
Biotin
Label protocol
Biotinylated cRNA were prepared according to the standard Affymetrix protocol from ~50ng total RNA (Expression Analysis Technical Manual, 2001, Affymetrix)
Hybridization protocol
Following fragmentation, ~50ng of cRNA were heated to 99˚C for 5 minutes, then heated at 45˚C for 5 minutes, prior to hybridization via constant agitation at 60rpm for 16 hours at 48˚C on an Affymetrix 450 Fluidics Station.
Scan protocol
Genechips were scanned using the Affymetrix GCS 3,000 Scanner
Data processing
RMA algorithm. Data was processed with Affymetrix Genechip command console software and analyzed using Affymetric Transcriptome Analysis Concole whereby probesets were differentially expressed at a fold-change of ≤ -2 or ≥ 2, ≥ 50% of samples have a detectable ablove background value of < 0.05 and a false discovery rate of < 0.05.
