Transcript profiles of some developmentally important genes detected in bovine oocytes and in vitro-produced blastocysts using RNA amplification and cDNA microarrays.
To study the mRNA transcript profiles of some potential candidate developmental genes during bovine oocyte and blastocyst stages, RNA amplification procedures, cDNA microarray of 82 target genes spotted onto glass slide and real-time polymerase chain reaction (PCR) were used. Messenger RNAs were isolated from in vitro-produced bovine matured oocytes and blastocysts. Using equal amounts of input mRNAs but different cycles of amplifications, cDNAs were produced and served as template for RNA amplification by the in vitro transcriptions. After amplification, the RNA yields transcribed from cDNAs of different cycles were evaluated both by hybridization on the cDNA microarrays and by using real-time PCR techniques. The analyses indicated best results from lower amplification cycle templates with consistent signals at hybridization. Generally, the RNA yield was directly proportional to the amplification cycle but inversely related with signal consistency at repeated hybridizations. Using the protocols established, equal amounts of amplified RNA from matured oocytes and blastocysts were hybridized to the array. Analyses of replicated hybridizations indicated that 35 transcripts were differentially expressed. Most of these were not described in previous bovine embryo studies. Independent analyses of 23 transcripts with real-time PCR and unamplified RNA confirmed the results of 22 genes. Moreover, the functional analyses showed various roles related to development. Hence, it is possible to conclude that the genes identified here are potential candidates for characterizing developmental competence, and that the methods established can be used for large-scale gene expression analysis with more comprehensive arrays.
Mamo
S
S
Institute of Animal Breeding Sciences, University of Bonn, Bonn, Germany.
Sargent
C A
CA
Affara
N A
NA
Tesfaye
D
D
El-Halawany
N
N
Wimmers
K
K
Gilles
M
M
Schellander
K
K
Ponsuksili
S
S
D000818:Animals; D001483:Base Sequence; D001755:Blastocyst / Q000378:metabolism*; D002417:Cattle / Q000196:embryology* / Q000235:genetics / Q000378:metabolism; D004622:Embryo, Mammalian / Q000378:metabolism; D005260:Female; D005784:Gene Amplification; D020869:Gene Expression Profiling / Q000662:veterinary; D020411:Oligonucleotide Array Sequence Analysis; D009865:Oocytes / Q000378:metabolism*; D016133:Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary; D012333:RNA, Messenger / Q000378:metabolism*; D014158:Transcription, Genetic
D012333:RNA, Messenger