The RNA Atlas expands the catalog of human non-coding RNAs.

Existing compendia of non-coding RNA (ncRNA) are incomplete, in part because they are derived almost exclusively from small and polyadenylated RNAs. Here we present a more comprehensive atlas of the human transcriptome, which includes small and polyA RNA as well as total RNA from 300 human tissues and cell lines. We report thousands of previously uncharacterized RNAs, increasing the number of documented ncRNAs by approximately 8%. To infer functional regulation by known and newly characterized ncRNAs, we exploited pre-mRNA abundance estimates from total RNA sequencing, revealing 316 microRNAs and 3,310 long non-coding RNAs with multiple lines of evidence for roles in regulating protein-coding genes and pathways. Our study both refines and expands the current catalog of human ncRNAs and their regulatory interactions. All data, analyses and results are available for download and interrogation in the R2 web portal, serving as a basis for future exploration of RNA biology and function.

Messenger RNA capture sequencing of extracellular RNA from human biofluids using a comprehensive set of spike-in controls.

Comprehensive transcriptome analysis of extracellular RNA (exRNA) purified from human biofluids is challenging because of the low RNA concentration and compromised RNA integrity. Here, we describe an optimized workflow to (1) isolate exRNA from different types of biofluids and (2) to prepare messenger RNA (mRNA)-enriched sequencing libraries using complementary hybridization probes. Importantly, the workflow includes 2 sets of synthetic spike-in RNA molecules as processing controls for RNA purification and sequencing library preparation and as an alternative data normalization strategy. For complete details on the use and execution of this protocol, please refer to Hulstaert et al. (2020).

Performance assessment of total RNA sequencing of human biofluids and extracellular vesicles.

RNA profiling has emerged as a powerful tool to investigate the biomarker potential of human biofluids. However, despite enormous interest in extracellular nucleic acids, RNA sequencing methods to quantify the total RNA content outside cells are rare. Here, we evaluate the performance of the SMARTer Stranded Total RNA-Seq method in human platelet-rich plasma, platelet-free plasma, urine, conditioned medium, and extracellular vesicles (EVs) from these biofluids. We found the method to be accurate, precise, compatible with low-input volumes and able to quantify a few thousand genes. We picked up distinct classes of RNA molecules, including mRNA, lncRNA, circRNA, miscRNA and pseudogenes. Notably, the read distribution and gene content drastically differ among biofluids. In conclusion, we are the first to show that the SMARTer method can be used for unbiased unraveling of the complete transcriptome of a wide range of biofluids and their extracellular vesicles.

Model-Based Classification for Digital PCR: Your Umbrella for Rain.

Standard data analysis pipelines for digital PCR estimate the concentration of a target nucleic acid by digitizing the end-point fluorescence of the parallel micro-PCR reactions, using an automated hard threshold. While it is known that misclassification has a major impact on the concentration estimate and substantially reduces accuracy, the uncertainty of this classification is typically ignored. We introduce a model-based clustering method to estimate the probability that the target is present (absent) in a partition conditional on its observed fluorescence and the distributional shape in no-template control samples. This methodology acknowledges the inherent uncertainty of the classification and provides a natural measure of precision, both at individual partition level and at the level of the global concentration. We illustrate our method on genetically modified organism, inhibition, dynamic range, and mutation detection experiments. We show that our method provides concentration estimates of similar accuracy or better than the current standard, along with a more realistic measure of precision. The individual partition probabilities and diagnostic density plots further allow for some quality control. An R implementation of our method, called Umbrella, is available, providing a more objective and automated data analysis procedure for absolute dPCR quantification.

Flexible analysis of digital PCR experiments using generalized linear mixed models.

The use of digital PCR for quantification of nucleic acids is rapidly growing. A major drawback remains the lack of flexible data analysis tools. Published analysis approaches are either tailored to specific problem settings or fail to take into account sources of variability. We propose the generalized linear mixed models framework as a flexible tool for analyzing a wide range of experiments. We also introduce a method for estimating reference gene stability to improve accuracy and precision of copy number and relative expression estimates. We demonstrate the usefulness of the methodology on a complex experimental setup.

Incidence and risk factors for pressure ulcers in the intensive care unit.

AIMS: To determine the incidence of pressure ulcers occurring at least 48 hours after admission and risk factors for pressure ulcers grade 2-4 in a long-stay surgical Intensive Care Unit (ICU) population. BACKGROUND: The incidence of pressure ulcers in intensive care units is larger than in non-intensive environments. DESIGN: Prospective descriptive research design. METHODS: Using pressure ulcers grade 2-4 as an outcome measure, a multivariate logistic regression analysis was used to identify the risk factors. Data were obtained on a daily basis in a surgical intensive care unit of the University Hospital Leuven between November 2003-March 2004. A total of 520 long-stay (>or= 24 hours) intensive care patients were included. RESULTS: Cumulative incidence of pressure ulcers grade 2-4 was 20.1%. The following variables were positively associated with pressure ulcers grade 2-4: history of vascular disease, treatment with Dopamine or Dobutamine, intermittent haemodialysis (IHD) or continuous veno-venous haemofiltration (CVVH), mechanical ventilation. Also preventive measures were statistically positively associated with pressure ulcers grade 2-4: turning, floating heels, alternating mattresses, adequate prevention. The use of sedatives, body temperature above 38.5 degrees C and sitting in chair where negatively associated with pressure ulcers. Pressure ulcers are statistically associated with different risk factors and preventive measures. CONCLUSION: The identified risk factors are eligible to be included in a new risk assessment scale for patients admitted to intensive care units. RELEVANCE TO CLINICAL PRACTICE: The novel insights have implications for risk assessment for patients in intensive care units. Patients admitted to intensive care units have other risk factors for pressure ulcers which are eligible to be included in a new risk assessment scale.