Non-radioisotopic quantitative RT-PCR to detect changes in mRNA levels during early mouse embryo development

Biochem Biophys Res Commun. 1993 Sep 15;195(2):769-75. doi: 10.1006/bbrc.1993.2112.

Abstract

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.

MeSH terms

  • Actins / biosynthesis*
  • Animals
  • Base Sequence
  • Blotting, Northern / methods
  • Bone Marrow / metabolism
  • Cell Line
  • Cyclins / biosynthesis
  • Embryo, Mammalian / physiology
  • Embryonic and Fetal Development*
  • Female
  • Macrophage Colony-Stimulating Factor / biosynthesis*
  • Mice
  • Mice, Inbred Strains
  • Molecular Sequence Data
  • Oligodeoxyribonucleotides
  • Oligonucleotides, Antisense
  • Polymerase Chain Reaction / methods*
  • Protein Biosynthesis
  • RNA, Messenger / analysis
  • RNA, Messenger / metabolism*
  • RNA-Directed DNA Polymerase
  • Receptors, Transferrin / biosynthesis
  • cdc25 Phosphatases

Substances

  • Actins
  • Cyclins
  • Oligodeoxyribonucleotides
  • Oligonucleotides, Antisense
  • RNA, Messenger
  • Receptors, Transferrin
  • Macrophage Colony-Stimulating Factor
  • RNA-Directed DNA Polymerase
  • cdc25 Phosphatases