Project description:Post-hybridization washing is an essential part of microarray experiments. Both, the quality of the experimental washing protocol and the adequate consideration of washing in intensity calibration ultimately affect the quality of the expression estimates extracted from the microarray intensities. We conducted experiments on GeneChip microarrays with altered protocols for washing, scanning and staining to study the probe-level intensity changes as a function of washing cycles. Particularly, three Affymetrix GeneChip HGU133plus2 arrays were hybridized and equilibrated for 16 hours in the hybridization oven. For one of the three arrays washing and staining was performed according to the manufacturer’s instructions. For another array the first scan was done immediately after low stringent wash and staining without intermitting stringent washing. Then, the array was stringently washed and scanned in alternating order three more times where each washing step consists of a definite number of washing cycles. The third array was low stringently washed followed by two stringent washing cycles and staining before the first scan. Subsequently it was analogously processed as array A. All three chips are repeatedly processed in a second series of alternating wash/scan-cycles which was performed using the same protocol for each chip as in the first series as described above. As in the first series the arrays were also stained a second time to compensate for any loss of bleached fluorescent dye. Analysis of the washing kinetics shows that the signal-to-noise ratio doubles roughly every ten stringent washing cycles. Washing can be characterized by time-dependent rate constants which reflect the heterogeneous character of target binding to microarray probes. We propose an empirical washing function which estimates the survival of probe bound targets. The washing function allows calibrating probe intensities for the effect of washing. On a relative scale, proper calibration for washing markedly increases expression measures especially in the limit of small and large values.
Project description:Household washing machines (WMs) launder soiled clothes and textiles, but do not sterilize them. We investigated the microbial exchange occurring in five household WMs. Samples from a new cotton T-shirt were laundered together with a normal laundry load. Analyses were performed on the influent water and the ingoing cotton samples, as well as the greywater and the washed cotton samples. The number of living bacteria was generally not lower in the WM e?uent water as compared to the influent water. The laundering process caused a microbial exchange of influent water bacteria, skin-, and clothes-related bacteria and biofilm-related bacteria in the WM. A variety of biofilm-producing bacteria were enriched in the e?uent after laundering, although their presence in the cotton sample was low. Nearly all bacterial genera detected on the initial cotton sample were still present in the washed cotton samples. A selection for typical skin- and clothes-related microbial species occurred in the cotton samples after laundering. Accordingly, malodour-causing microbial species might be further distributed to other clothes. The bacteria on the ingoing textiles contributed for a large part to the microbiome found in the textiles after laundering.
Project description:Riboswitches are structured allosteric RNA molecules that change conformation in response to a metabolite binding event, eventually triggering a regulatory response. Computational modelling of the structure of these molecules is complicated by a complex network of tertiary contacts, stabilized by the presence of their cognate metabolite. In this work, we focus on the aptamer domain of SAM-I riboswitches and show that Restricted Boltzmann machines (RBM), an unsupervised machine learning architecture, can capture intricate sequence dependencies induced by secondary and tertiary structure, as well as a switching mechanism between open and closed conformations. The RBM model is then used for the design of artificial allosteric SAM-I aptamers. To experimentally validate the functionality of the designed sequences, we resort to chemical probing (SHAPE-MaP), and develop a tailored analysis pipeline adequate for high-throughput tests of diverse homologous sequences. We probed a total of 476 RBM designed sequences in two experiments, showing between 20% and 40% divergence from any natural sequence, obtaining ≈ 30% success rate of correctly structured aptamers that undergo a structural switch in response to SAM.
Project description:The aims of this study were to determine the impact of different levels of RNA degradation as well as to ascertain if the gene expression profiles obtained from bladder washing correlates to that obtained from the related bladder tumor. Keywords: RNA degradation, bladder washing, bladder tumor, bladder cancer