Selection of normalizer genes in conducting relative gene expression analysis of embryos

Abstract

BACKGROUND In relative gene expression analysis, a reference gene for sample normalization is required for determining target expression changes among experimental treatment groups. Since some developmental toxicants secondarily cause general growth retardation and/or other general biological changes, commonly used housekeeping genes may not serve as accurate normalizers. METHODS We conducted real‐time polymerase chain reaction (PCR) with normalization to calculate relative target transcriptional change, using housekeeping and structure‐specific expression genes as normalizers. Relative levels of Hoxb1 expression were measured in cultured rodent embryos at 24 hr post retinoic acid (RA) administration. Transcriptional response was also evaluated using two novel compounds that produced posterior axial and growth defects in rat whole‐embryo culture. Embryos treated with these compounds were evaluated for general biological processes, and their respective biological states were considered in the context of the relative gene expression change calculated with the housekeeping normalizers. RESULTS Normalized RA‐induced Hoxb1 expression demonstrated that only some reference genes accurately quantitated the expected 1.5‐ to 2‐fold increase in Hoxb1 expression. Evaluation of the test compounds demonstrated that only normalization with the spatially‐restricted hindbrain gene, Krox‐20, calculated significant expression decreases of T‐gene, a gene known to be functionally relevant in posterior axial development. Reduction in T‐gene expression was confirmed qualitatively by whole‐mount in situ hybridization. CONCLUSIONS Prudent reference gene selection is important in evaluating relative gene expression in embryos. An experimental control design is proposed to facilitate the identification of normalizing genes that will accurately calculate relative gene expression change in treated embryos. Birth Defects Research (Part A), 2003. © 2003 Wiley‐Liss, Inc.

Comprehensive selection of reference genes for quantitative RT-PCR analysis of murine extramedullary hematopoiesis during development

“…Mamo et al [33] showed moderate changes for the genes ACTB , GAPDH , HPRT1 and others in an in vitro and in vivo experiment at different stages using whole embryos. The cells comprising the embryo have a vastly heterogeneous nature, which leads to greater variation in the endogenous biological processes, and greater variation in the sensitivity of cells to their environment [34]. Because these factors can introduce intra- and inter assay variations, we elected to isolate the specific EH organs of interest (heart, liver, spleen, thymus) and analyze gene expression in these organs independently instead of analyzing whole embryo homogenates, as has been previously described [35].…”

Quantitative evaluation and selection of reference genes in mouse oocytes and embryos cultured in vivo and in vitro

“…Moreover, embryonic samples have additional sources of variations. Unlike cell lines and single-organ tissues, the cells comprising the embryo have inherently a vastly heterogeneous nature, which leads to greater variation in the endogenous biological processes, and greater variation in the sensitivity of the cells to the treatment [16]. There is high probability that any of these factors can introduce intra- and inter assay variations for which normalization is required.…”

Developmental expression profiles of Xenopus laevis reference genes

“…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).…”