Cell properties depend mainly on the nucleic acid and protein content. The mRNA molecules represent a connection between DNA and protein. The spatial and temporal gene expression changes are a key mechanism in cell differentiation. Two different types of Xenopus mRNAs can be distinguished: (1) the maternal mRNAs, which occur in the oocyte before fertilization and originate from the female, and (2) the zygotic mRNAs, newly synthesized mainly after the midblastula transition (MBT). The correct spatial and temporal expression of both types of mRNAs is necessary for the first developmental processes such as the main body axis formation, gastrulation, germ layers induction, and others. The mRNA expression has been broadly studied by common methods such as Northern blotting, RNase protection assays, in situ hybridization, reverse transcription-polymerase chain reaction (RT-PCR), and microarray analysis. In the last decade, a new highly sensitive and specific method for RNA/DNA quantification, the real-time RT-PCR (qPCR), was introduced. The sensitivity, dynamic range, linearity of the measurements, and robustness make the qPCR a method of choice for the quantitative analysis of mRNA expression. For these reasons, it is also frequently used as an independent validation tool for the verification of microarray expression data (Giulietti et al.,2001).
The quantification can be carried out by two different approaches. The absolute method determines the number of mRNA copies in the sample from a calibration curve obtained from samples of cDNA complementary to mRNA of known concentrations. The relative approach compares copies of the target mRNA with those of a reference gene. The metabolic, structural, and ribosomal RNA genes, such as those coding for beta-actin, GAPDH, beta-tubulin, 18S rRNA, and so on, belong among the most popular reference genes (Suzuki et al.,2000). The normalisation against a reference gene requires a constant mRNA expression of the reference gene, which would not vary during the cell cycle, in different cell types, or during development. Moreover, the expression level of the reference gene should be similar to that of the target gene. However, several recent studies have shown that, in vertebrate systems, the mRNA expression of reference genes differed among tissues, various cells, and after treatment (Zhang,2003; Bas et al.,2004; de Kok et al.,2004; Bustin and Nolan,2004; Vandesompele et al.,2002; Brunner et al.,2004; Radonic et al.,2004). To date, the most reliable method for normalisation appears to relate the mRNA data to the total RNA content of the sample preparation subjected to the reverse transcription reaction (Bustin,2002). The most important step in the normalisation to total RNA is thus a precise determination of RNA concentration and the quality of RNA preparation (Fig. 1). Xenopus laevis mRNA expression studies have almost always used the genes coding for elongation factor eEF-1 alpha, ornithine decarboxylase (ODC), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), L8-ribosomal protein, and H4-histone protein as reference genes (Chang,2004; Veldholen et al.,2002; Wardle and Smith,2004). The aim of this study was to determine, by qPCR and normalisation to total RNA content, the mRNA expression profiles of the above-mentioned five reference genes, and, of two other genes, coding for N-tubulin and Xbra, in the course of X. laevis early development. The profiles are stage- and gene-specific and allow to determine stages at which the normalisation with the reference genes appears to be plausible and when it would give more or less biased results.