Real-time reverse transcription-polymerase chain reaction analysis of translation initiation factor 1A (eIF-1A) in human and mouse preimplantation embryos.
Fluorescence-monitored real-time reverse transcription-polymerase chain reaction (RT-PCR) was used to study steady state concentrations of translation initiation factor eIF-1A mRNA in mouse and human preimplantation embryos. Its expression in human embryos has not been described previously. Human oocytes, and 2-cell and 4-cell embryos all showed comparable total concentrations of eIF-1A RNA, indicating a gradual decrease in the average concentration per blastomere during these developmental stages. A 4-fold increase was observed in the 8-cell embryos. This concentration remained at the morula stage, followed by a 7- to 8-fold further increase at the blastocyst stage. Mouse preimplantation embryos already showed increased concentrations of eIF-1A RNA at the 2-cell stage. Thus, transcription levels of the eIF-1A gene are associated with embryonic gene activation (EGA) in both species. The method used, real time RT-PCR, proved to be sensitive enough to detect quantitative expression in single mouse blastomeres, the observed values for steady-state concentrations of mRNA in single blastomeres correlating well with the values for whole embryos. The possibility to study gene expression quantitatively in single blastomeres may be useful in preimplantation genetic diagnosis.
Lindeberg
M
M
Clinical Research Centre, Karolinska Institute Novum, Karolinska University Hospital, 14186 Stockholm, Sweden. maria.lindeberg@crc.ki.se
Hovatta
O
O
Ahrlund-Richter
L
L
D000818:Animals; D001755:Blastocyst / Q000378:metabolism*; D017931:DNA Primers / Q000737:chemistry; D018076:DNA, Complementary / Q000378:metabolism; D004996:Ethidium / Q000494:pharmacology; D015977:Eukaryotic Initiation Factor-1 / Q000096:biosynthesis* / Q000737:chemistry*; D005260:Female; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D012313:RNA / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014158:Transcription, Genetic
D017931:DNA Primers; D018076:DNA, Complementary; D015977:Eukaryotic Initiation Factor-1; D012333:RNA, Messenger; C026022:eukaryotic peptide initiation factor-1A; D012313:RNA; D004996:Ethidium