Optimization of real time RT‐PCR methods for the analysis of gene expression in mouse eggs and preimplantation embryos

Abstract

This study was carried out to optimize conditions for using real time RT-PCR as an efficient and precise quantitative method for estimating the transcript levels of genes expressed in samples containing miniscule amounts of RNA, such as single mammalian oocytes and embryos. First, using mouse eggs and blastocysts, we tested three kinds of RNA isolation or collection methods: TRIZOL reagent, oligo-dT conjugated beads, or three freeze/thaw cycles with the reverse transcription buffer. There were no significant differences among three groups in mRNA quantity as assayed by real time RT-PCR analysis. Second, we compared the efficacy of real time analysis between TaqMan fluorescent probes and the SYBR-green dye system. The two systems presented similar real time RT-PCR profiles for the 16s ribosomal protein gene from oocytes to blastocysts. Third, RNA from mouse embryos at defined stages of preimplantation development were isolated and the levels of transcripts encoded by several housekeeping genes (GAPDH, beta-actin, ribosomal protein L7, 16s ribosomal protein, histone H2A.Z) were quantitatively analyzed by real time RT-PCR. The histone H2A.Z and 16s ribosomal protein slightly increased from the egg to blastocyst stages by approximately 10- and 30-fold, respectively. However, other transcripts increased more than 300-fold as a function of developmental stage from eggs to blastocysts. Our results suggest that the simple freezing/thawing method for RNA collection, the economic SYBR-green dye system, and histone H2A.Z gene as an internal control should be useful for the real time RT-PCR analysis of single mouse eggs and preimplantation embryos.

Characterization of mitochondrial replication and transcription control during rat early development in vivo and in vitro

“…This expression pattern agrees with that in in vivo mouse Mitochondrial replication/transcription in rat ova embryos , in vitro mouse embryos (Jeong et al 2005) and in vitro-produced bovine embryos (Robert et al 2002). The expression of Gapd could be a useful internal control not to compare between stages but for fixed stages.…”

Delayed and stage specific phosphorylation of H2AX during preimplantation development of γ-irradiated mouse embryos

“…In fact, early embryonic development of Xenopus is punctuated by so called midblastula transition in which when a burst of transcription and prolonged cell cycle time take place (Newport & Kirschner 1982). Similar midblastula transition exists in mice and a surge of transcription was reported to occur at the morula stage (Jeong et al 2005). It is interesting to note that the morula is the stage where mouse embryos start to show cleavage delay when fertilized by irradiated sperm (Adiga, unpublished observation).…”

p21 provides stage specific DNA damage control to preimplantation embryos

“…Since p21 plays an important role in G2/ M arrest, the expression of p21 was examined in control embryos, 3 Gy directly irradiated embryos and spermirradiated embryos with the use of H2A.Z as an internal standard for the real time reverse transcriptasepolymerase chain reaction (RT-PCR) analyses (Jeong et al, 2005). The results are shown in Figure 3.…”

“…Xenopus embryogenesis has been characterized by a period called midblastula transition, in which a burst of transcription takes place concomitant with prolongation of cell cycle time (Newport and Kirschner, 1982). In mice, the midblastula transition period corresponds to the morulablastocyst stage with a similar surge of transcription of p21 provides stage specific DNA damage control SK Adiga et al housekeeping genes (Jeong et al, 2005). Indeed, the increase in the level of expression of b-actin gene was more than 200-fold, while that of H2A.Z gene less than 10-fold, making this gene particularly suitable as the internal standard for RT-PCR analyses.…”