Non-radioisotopic quantitative RT-PCR to detect changes in mRNA levels during early mouse embryo development.
We developed a non-radioisotopic quantitative RT-PCR method with high sensitivity and reproducibility. The results of this RT-PCR were in agreement with those of the Northern blot analysis. We measured the mRNA levels of beta-actin, transferrin receptor, and two cell cycle-related genes, cyclin B and cdc25, in early mouse embryos by the RT-PCR. In late two-cell stage embryos, beta-actin, transferrin receptor and cyclin B mRNA levels were 10-20% of those in MII stage oocytes. In contrast, the cdc25 mRNA levels were not different between these stages. When we cultured mouse embryos, the presence of an RNA polymerase inhibitor, alpha-amanitin, in the medium did not affect the mRNA levels at the two-cell stage, indicating that most of the detected mRNAs in two-cell embryos were maternally derived. These results suggest that the rate of mRNA degradation is different between cyclin B and cdc25 during early embryogenesis.
Yokoi
H
H
Department of Clinical Molecular Biology, Faculty of Medicine, Kyoto University, Japan.
Natsuyama
S
S
Iwai
M
M
Noda
Y
Y
Mori
T
T
Mori
K J
KJ
Fujita
K
K
Nakayama
H
H
Fujita
J
J
D000199:Actins / Q000096:biosynthesis*; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern / Q000379:methods; D001853:Bone Marrow / Q000378:metabolism; D002460:Cell Line; D016213:Cyclins / Q000096:biosynthesis; D004622:Embryo, Mammalian / Q000502:physiology; D005314:Embryonic and Fetal Development; D005260:Female; D016173:Macrophage Colony-Stimulating Factor / Q000096:biosynthesis*; D051379:Mice; D008815:Mice, Inbred Strains; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D016376:Oligonucleotides, Antisense; D016133:Polymerase Chain Reaction / Q000379:methods*; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000032:analysis / Q000378:metabolism*; D012194:RNA-Directed DNA Polymerase; D011990:Receptors, Transferrin / Q000096:biosynthesis; D020687:cdc25 Phosphatases
D000199:Actins; D016213:Cyclins; D009838:Oligodeoxyribonucleotides; D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger; D011990:Receptors, Transferrin; D016173:Macrophage Colony-Stimulating Factor; D012194:RNA-Directed DNA Polymerase; D020687:cdc25 Phosphatases