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Nuclear receptors (NRs) belong to a superfamily of transcription factors that regulate numerous homeostatic, metabolic and reproductive processes. Taken together with their modulation by small lipophilic molecules, they also represent an important and successful class of drug targets. Although many NRs have been targeted successfully, the majority have not, and one third are still orphans. Here we report the development of an in vivo GFP-based reporter system suitable for monitoring NR activities in all cells and tissues using live zebrafish (Danio rerio). The human NR fusion proteins used also contain a new affinity tag cassette allowing the purification of receptors with bound molecules from responsive tissues. We show that these constructs 1) respond as expected to endogenous zebrafish hormones and cofactors, 2) facilitate efficient receptor and cofactor purification, 3) respond robustly to NR hormones and drugs and 4) yield readily quantifiable signals. Transgenic lines representing the majority of human NRs have been established and are available for the investigation of tissue- and isoform-specific ligands and cofactors.

High-throughput sequencing is increasingly favoured to assay the presence and abundance of microRNAs (miRNAs) in biological samples, even from low RNA amounts, and a number of commercial vendors now offer kits that allow miRNA sequencing from sub-nanogram (ng) inputs. Although biases introduced during library preparation have been documented, the relative performance of current reagent kits has not been investigated in detail. Here, six commercial kits capable of handling <100ng total RNA input were used for library preparation, performed by kit manufactures, on synthetic miRNAs of known quantities and human total RNA samples. We compared the performance of miRNA detection sensitivity, reliability, titration response and the ability to detect differentially expressed miRNAs. In addition, we assessed the use of unique molecular identifiers (UMI) sequence tags in one kit. We observed differences in detection sensitivity and ability to identify differentially expressed miRNAs between the kits, but none were able to detect the full repertoire of synthetic miRNAs. The reliability within the replicates of all kits was good, while larger differences were observed between the kits, although none could accurately quantify the relative levels of the majority of miRNAs. UMI tags, at least within the input ranges tested, offered little advantage to improve data utility. In conclusion, biases in miRNA abundance are heavily influenced by the kit used for library preparation, suggesting that comparisons of datasets prepared by different procedures should be made with caution. This article is intended to assist researchers select the most appropriate kit for their experimental conditions.

Vitamin D has attracted much attention by its ability to stop cell proliferation and induce differentiation, which became of particular interest for the treatment of cancer and psoriasis. We performed an expression profile of 12 hours and 24 hours 1alpha,25-dihydroxyvitamin D(3) (1alpha,25(OH)(2)D(3)) treated primary human keratinocytes, to determine the changes in gene expression induced by the steroid in order to improve our understanding of the biological activity of 1alpha,25(OH)(2)D(3). This we expect to be useful for establishing a test system for vitamin D analogs or might open new therapeutic targets or uses for the hormone. For the filter array experiments a non-redundant set of 2135 sequence verified EST clones was used. The normalized raw data of 2 filters per time point were combined and subjected to SAM analysis to further increase the statistical significance. 86 positive and 50 negative genes were identified after 12 h. The numbers went down to 43 positive and 1 negative gene after 24 h of treatment. Fifteen genes are up-regulated over a longer period of time (12 h and 24 h). Results were verified by real-time PCR and/or Northern blots. Targets identified are involved in intracellular signaling, transcription, cell cycle, metabolism, cellular growth, constitution of the extracellular matrix or the cytoskeleton and apoptosis, immune responses, and DNA repair, respectively. Expression profiles showed an initial stop of proliferation and induction of differentiation, and resumed proliferation after prolonged incubation, most likely due to degradation of the hormone.