Normal gene expression variation in Enchytraeus albidus - transcriptional responses to control conditions

Several questions remain on how ecologically relevant can a change in gene expression be or how to quantify after which changes are beyond normal biological variation. Knowing genes are highly sensitive and self-regulating pieces can we design our experiments to minimize natural variability sources? E.g., are results from experiments performed at different times always comparable? Further, the effect of carriers (solvents) is often neglected. The main aim of the present paper was to create critical discussion on these and other questions, while providing suggestions for improvement and interpretation. We have analysed gene data sets comparing standard control test conditions: 1) from similar experiments performed at different occasions in time; 2) to culture media, 3) to acetone control, 4) from various exposure periods (2, 4 and 8 days) and 5) between replicates. In general, the transference of organisms from culture media to standard testing suggests a lowered general metabolism. Responses to acetone seem to be related with changes in soil structure, usually associated with hypoxia conditions. Coefficient of variance (CV) between biological replicates was >0.1 for 17% of the genes being as high as 1.3. These results represent the first contribution for the establishment of a normal operating range (NOR) for E. albidus gene expression. Further, comparison between various control exposure times showed significant variation - organisms have a “time-like” transcription signature. Tests may be designed to potentially reduce these variability sources, although, this will partly be inherent to the species, and most important is to understand the ground for interpretations. Gene expression in E.albidus was measured in several control samples. The experimental design consisted in three analyses to infer: 1) the effect of transferring animals from culture conditions onto standard control test conditions: control (CT) versus culture (CULT); 2) the effect of acetone (frequently used as carrier) compared to water: acetone (ACT) versus water control (CT); 3) the effect of different exposure time in CT conditions: CT4d and CT8d vesus CT2d