A new set of endogenous control genes for use in quantitative real-time PCR experiments show that formin Ldia2dex transcripts are enriched in the early pond snail embryo

Although the pond snail Lymnaea stagnalis is an emerging model organism for molecular studies in a wide variety of fields, there are a limited number of verified endogenous control genes for use in quantitative real-time PCR (qRT-PCR). As part of larger study on snail chirality, or left-right asymmetry, we wished to assay gene expression in pond snail embryos. We therefore evaluated six candidate control genes, by comparing their expression in three tissues (ovotestis, foot, and embryo) and across three programs (geNorm, Normfinder and Bestkeeper). The specific utility of these control genes was then tested by investigating the relative expression of six experimental transcripts, including the formin Ldia2, a gene that has been associated with chiral variation in L. stagnalis. All six control genes were found to be suitable for use in the three tissues tested. Of the six experimental genes, it was found that all were relatively depleted in the early embryo compared with other tissues, except the formin gene Ldia2. Instead, transcripts of the wild type Ldia2dex were enriched in the embryo, whereas a non-functional frameshifted version Ldia2sin was severely depleted. These differences in Ldia2sin expression were less evident in the ovotestis and not evident in the foot tissue, possibly because nonsense-mediated decay is obscured in actively transcribing tissues. This work therefore provides a set of control genes that may be useful to the wider community, and illustrates how they may be used to assay differences in expression in a variety of tissues.