title: Monitoring gene expression in a single Xenopus oocyte using multiple cytoplasmic collections and quantitative RT-PCR.
abstract: Oocytes and eggs of the African clawed frog, Xenopus laevis, are commonly used in gene expression studies. However, monitoring transcript levels in the individual living oocytes remains challenging. To address this challenge, we used a technique based on multiple repeated collections of nanoliter volumes of cytoplasmic material from a single oocyte. Transcript quantification was performed by quantitative RT-PCR. The technique allowed monitoring of heterologous gene expression in a single oocyte without affecting its viability. We also used this approach to profile the expression of endogenous genes in living Xenopus oocytes. Although frog oocytes are traditionally viewed as a homogenous cell population, a significant degree of gene expression variation was observed among the individual oocytes. A lognormal distribution of transcript levels was revealed in the oocyte population. Finally, using this technique, we observed a dramatic decrease in the content of various cytoplasmic mRNAs in aging unfertilized eggs but not in oocytes, suggesting a link between mRNA degradation and egg apoptosis.
authors.affiliation: Research Center for Environmental Genomics, Kobe University, Japan.
authors.forename: Alexander A
authors.initials: AA
authors.lastname: Tokmakov
authors.forename: Takanori
authors.initials: T
authors.lastname: Hashimoto
authors.forename: Yushi
authors.initials: Y
authors.lastname: Hasegawa
authors.forename: Sho
authors.initials: S
authors.lastname: Iguchi
authors.forename: Tetsushi
authors.initials: T
authors.lastname: Iwasaki
authors.forename: Yasuo
authors.initials: Y
authors.lastname: Fukami
mesh_terms: D000818:Animals; D015153:Blotting, Western; D003593:Cytoplasm / Q000378:metabolism*; D005260:Female; D020869:Gene Expression Profiling; D018507:Gene Expression Regulation, Developmental; D008156:Luciferases / Q000378:metabolism; D009865:Oocytes / Q000166:cytology / Q000378:metabolism*; D010063:Ovum / Q000166:cytology / Q000378:metabolism*; D020871:RNA Stability / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014982:Xenopus laevis
chemical_list: D012333:RNA, Messenger; D008156:Luciferases
keywords: Xenopus oocytes; gene expression variation; mRNA degradation; quantitative RT-PCR; single-cell gene expression analysis