Showing papers by "Jo Vandesompele published in 2021"

Closing the circle: current state and perspectives of circular RNA databases.

Abstract: Circular RNAs (circRNAs) are covalently closed RNA molecules that have been linked to various diseases, including cancer. However, a precise function and working mechanism are lacking for the larger majority. Following many different experimental and computational approaches to identify circRNAs, multiple circRNA databases were developed as well. Unfortunately, there are several major issues with the current circRNA databases, which substantially hamper progression in the field. First, as the overlap in content is limited, a true reference set of circRNAs is lacking. This results from the low abundance and highly specific expression of circRNAs, and varying sequencing methods, data-analysis pipelines, and circRNA detection tools. A second major issue is the use of ambiguous nomenclature. Thus, redundant or even conflicting names for circRNAs across different databases contribute to the reproducibility crisis. Third, circRNA databases, in essence, rely on the position of the circRNA back-splice junction, whereas alternative splicing could result in circRNAs with different length and sequence. To uniquely identify a circRNA molecule, the full circular sequence is required. Fourth, circRNA databases annotate circRNAs' microRNA binding and protein-coding potential, but these annotations are generally based on presumed circRNA sequences. Finally, several databases are not regularly updated, contain incomplete data or suffer from connectivity issues. In this review, we present a comprehensive overview of the current circRNA databases and their content, features, and usability. In addition to discussing the current issues regarding circRNA databases, we come with important suggestions to streamline further research in this growing field.

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

Abstract: 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.

The dangers of using Cq to quantify nucleic acid in biological samples; a lesson from COVID-19.

Abstract: Background SARS-CoV-2 RNA quantities, measured by reverse transcription quantitative PCR (RT-qPCR), have been proposed to stratify clinical risk or determine analytical performance targets. We investigated reproducibility and how setting diagnostic cut-offs altered the clinical sensitivity of COVID-19 testing. Methods Quantitative SARS-CoV-2 RNA distributions (Cq and copies/mL) from more than 6000 patients from three clinical laboratories in UK, Belgium and the Republic of Korea were analyzed. Impact of Cq cut-offs on clinical sensitivity was assessed. The June/July 2020 INSTAND EQA scheme SARS-CoV-2 materials were used to estimate laboratory reported copies/mL and to estimate the variation in copies/mL for a given Cq. Results When the WHO suggested Cq cut-off of 25 was applied, the clinical sensitivity dropped to as little as about 16%. Clinical sensitivity also dropped to as little as about 27% when a simulated LOD of 106 copies/mL was applied. The inter-laboratory variation for a given Cq value was >1000 fold in copies/mL (99% CI). Conclusion While RT-qPCR has been instrumental in the response to COVID-19, we recommend Cq (Ct or Cp) values not be used to set clinical cut-offs, or diagnostic performance targets, due to poor inter-laboratory reproducibility; calibrated copy-based units (used elsewhere in virology) offer more reproducible alternatives. We also report a phenomenon where diagnostic performance may change relative to the effective reproduction number (R). Our findings indicate that the disparities between patient populations across time are an important consideration when evaluating or deploying diagnostic tests. This is especially relevant to the emergency situation of an evolving pandemic.

Minimally invasive classification of paediatric solid tumours using reduced representation bisulphite sequencing of cell-free DNA: a proof-of-principle study

Abstract: In the clinical management of paediatric solid tumours, histological examination of tumour tissue obtained by a biopsy remains the gold standard to establish a conclusive pathological diagnosis. The DNA methylation pattern of a tumour is known to correlate with the histopathological diagnosis across cancer types and is showing promise in the diagnostic workup of tumour samples. This methylation pattern can be detected in the cell-free DNA. Here, we provide proof-of-concept of histopathologic classification of paediatric tumours using cell-free reduced representation bisulphite sequencing (cf-RRBS) from retrospectively collected plasma and cerebrospinal fluid samples. We determined the correct tumour type in 49 out of 60 (81.6%) samples starting from minute amounts (less than 10 ng) of cell-free DNA. We demonstrate that the majority of misclassifications were associated with sample quality and not with the extent of disease. Our approach has the potential to help tackle some of the remaining diagnostic challenges in paediatric oncology in a cost-effective and minimally invasive manner.

MISpheroID: a knowledgebase and transparency tool for minimum information in spheroid identity

Abstract: Spheroids are three-dimensional cellular models with widespread basic and translational application across academia and industry. However, methodological transparency and guidelines for spheroid research have not yet been established. The MISpheroID Consortium developed a crowdsourcing knowledgebase that assembles the experimental parameters of 3,058 published spheroid-related experiments. Interrogation of this knowledgebase identified heterogeneity in the methodological setup of spheroids. Empirical evaluation and interlaboratory validation of selected variations in spheroid methodology revealed diverse impacts on spheroid metrics. To facilitate interpretation, stimulate transparency and increase awareness, the Consortium defines the MISpheroID string, a minimum set of experimental parameters required to report spheroid research. Thus, MISpheroID combines a valuable resource and a tool for three-dimensional cellular models to mine experimental parameters and to improve reproducibility.

Targeted Therapy of TERT-Rearranged Neuroblastoma with BET Bromodomain Inhibitor and Proteasome Inhibitor Combination Therapy

Abstract: Purpose: TERT gene rearrangement with transcriptional superenhancers leads to TERT overexpression and neuroblastoma. No targeted therapy is available for clinical trials in patients with TERT-rearranged neuroblastoma. Experimental Design: Anticancer agents exerting the best synergistic anticancer effects with BET bromodomain inhibitors were identified by screening an FDA-approved oncology drug library. The synergistic effects of the BET bromodomain inhibitor OTX015 and the proteasome inhibitor carfilzomib were examined by immunoblot and flow cytometry analysis. The anticancer efficacy of OTX015 and carfilzomib combination therapy was investigated in mice xenografted with TERT-rearranged neuroblastoma cell lines or patient-derived xenograft (PDX) tumor cells, and the role of TERT reduction in the anticancer efficacy was examined through rescue experiments in mice. Results: The BET bromodomain protein BRD4 promoted TERT-rearranged neuroblastoma cell proliferation through upregulating TERT expression. Screening of an approved oncology drug library identified the proteasome inhibitor carfilzomib as the agent exerting the best synergistic anticancer effects with BET bromodomain inhibitors including OTX015. OTX015 and carfilzomib synergistically reduced TERT protein expression, induced endoplasmic reticulum stress, and induced TERT-rearranged neuroblastoma cell apoptosis which was blocked by TERT overexpression and endoplasmic reticulum stress antagonists. In mice xenografted with TERT-rearranged neuroblastoma cell lines or PDX tumor cells, OTX015 and carfilzomib synergistically blocked TERT expression, induced tumor cell apoptosis, suppressed tumor progression, and improved mouse survival, which was largely reversed by forced TERT overexpression. Conclusions: OTX015 and carfilzomib combination therapy is likely to be translated into the first clinical trial of a targeted therapy in patients with TERT-rearranged neuroblastoma.

Robust sequential biophysical fractionation of blood plasma to study variations in the biomolecular landscape of systemically circulating extracellular vesicles across clinical conditions.

Abstract: Separating extracellular vesicles (EV) from blood plasma is challenging and complicates their biological understanding and biomarker development. In this study, we fractionate blood plasma by combining size-exclusion chromatography (SEC) and OptiPrep density gradient centrifugation to study clinical context-dependent and time-dependent variations in the biomolecular landscape of systemically circulating EV. Using pooled blood plasma samples from breast cancer patients, we first demonstrate the technical repeatability of blood plasma fractionation. Using serial blood plasma samples from HIV and ovarian cancer patients (n = 10) we next show that EV carry a clinical context-dependent and/or time-dependent protein and small RNA composition, including miRNA and tRNA. In addition, differential analysis of blood plasma fractions provides a catalogue of putative proteins not associated with systemically circulating EV. In conclusion, the implementation of blood plasma fractionation allows to advance the biological understanding and biomarker development of systemically circulating EV.

Circulating RNA biomarkers in diffuse large B-cell lymphoma: a systematic review

Abstract: Diffuse large B-cell lymphoma (DLBCL) is the most common histological subtype of non-Hodgkin’s lymphomas (NHL). DLBCL is an aggressive malignancy that displays a great heterogeneity in terms of morphology, genetics and biological behavior. While a sustained complete remission is obtained in the majority of patients with standard immunochemotherapy, patients with refractory of relapsed disease after first-line treatment have a poor prognosis. This patient group represents an important unmet need in lymphoma treatment. In recent years, improved understanding of the underlying molecular pathogenesis had led to new classification and prognostication tools, including the development of cell-free biomarkers in liquid biopsies. Although the majority of studies have focused on the use of cell-free fragments of DNA (cfDNA), there has been an increased interest in circulating-free coding and non-coding RNA, including messenger RNA (mRNA), microRNA (miRNA), long non-coding RNA (lncRNA) and circular RNA (circRNA), as well as RNA encapsulated in extracellular vesicles or tumor-educated platelets (TEPs). We performed a systematic search in PubMed to identify articles that evaluated circulating RNA as diagnostic, subtype, treatment response or prognostic biomarkers in a human DLBCL population. A total of 35 articles met the inclusion criteria. The aim of this systematic review is to present the current understanding of circulating RNA molecules as biomarker in DLBCL and to discuss their future potential.

The long non-coding RNA SAMMSON is essential for uveal melanoma cell survival

Abstract: Long non-coding RNAs (lncRNAs) can exhibit cell-type and cancer-type specific expression profiles, making them highly attractive as therapeutic targets. Pan-cancer RNA sequencing data revealed broad expression of the SAMMSON lncRNA in uveal melanoma (UM), the most common primary intraocular malignancy in adults. Currently, there are no effective treatments for UM patients with metastatic disease, resulting in a median survival time of 6-12 months. We aimed to investigate the therapeutic potential of SAMMSON inhibition in UM. Antisense oligonucleotide (ASO)-mediated SAMMSON inhibition impaired the growth and viability of a genetically diverse panel of uveal melanoma cell lines. These effects were accompanied by an induction of apoptosis and were recapitulated in two uveal melanoma patient derived xenograft (PDX) models through subcutaneous ASO delivery. SAMMSON pulldown revealed several candidate interaction partners, including various proteins involved in mitochondrial translation. Consequently, inhibition of SAMMSON impaired global, mitochondrial and cytosolic protein translation levels and mitochondrial function in uveal melanoma cells. The present study demonstrates that SAMMSON expression is essential for uveal melanoma cell survival. ASO-mediated silencing of SAMMSON may provide an effective treatment strategy to treat primary and metastatic uveal melanoma patients.

Genome-wide study of the effect of blood collection tubes on the cell-free DNA methylome

Abstract: The methylation pattern of cfDNA, isolated from liquid biopsies, is gaining substantial interest for diagnosis and monitoring of diseases. We have evaluated the impact of type of blood collection t...

Validation of Circular RNAs Using RT-qPCR After Effective Removal of Linear RNAs by Ribonuclease R

Abstract: Circular RNAs (circRNAs) are a class of endogenous noncoding RNAs that have been shown to play a role in normal development, homeostasis, and disease, including cancer. CircRNAs are formed through a process called back-splicing, which results in a covalently closed loop with a nonlinear back-spliced junction (BSJ). In general, circRNA BSJs are predicted in RNA sequencing data using one of numerous circRNA detection algorithms. Selected circRNAs are then typically validated using an orthogonal method such as reverse transcription quantitative PCR (RT-qPCR) with circRNA-specific primers. However, linear transcripts originating from endogenous trans-splicing can lead to false-positive signals both in RNA sequencing and in RT-qPCR experiments. Therefore, it is essential to perform the RT-qPCR validation step only after linear RNAs have been degraded using an exonuclease such as ribonuclease R (RNase R). Several RNase R protocols are available for circRNA detection using RNA sequencing or RT-qPCR. These protocols-which vary in enzyme concentration, RNA input amount, incubation times, and cleanup steps-typically lack a detailed validated standard protocol and fail to provide a range of conditions that deliver accurate results. As such, some protocols use RNase R concentrations that are too high, resulting in partial degradation of the target circRNAs. Here, we describe an optimized workflow for circRNA validation, combining RNase R treatment and RT-qPCR. First, we outline the steps for circRNA primer design and qPCR assay validation. Then, we describe RNase R treatment of total RNA and, importantly, a subsequent essential buffer cleanup step. Lastly, we outline the steps to perform the RT-qPCR and discuss the downstream data analyses. © 2021 Wiley Periodicals LLC. Basic Protocol 1: CircRNA primer design and qPCR assay validation Basic Protocol 2: RNase R treatment, cleanup, and RT-qPCR.

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

Abstract: Summary 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 of RNA purification kits and blood collection tubes in the Extracellular RNA Quality Control (exRNAQC) study

Abstract: The use of blood-based extracellular RNA (exRNA) as clinical biomarker requires the implementation of a validated procedure for sample collection, processing and profiling. So far, no study has systematically addressed the pre-analytical variables affecting transcriptome analysis of exRNAs. In the exRNAQC study, we evaluated 10 blood collection tubes, 3 time points between blood draw and downstream processing, and 8 RNA purification methods using the supplier-specified minimum and maximum biofluid input volumes. The impact of these pre-analytics is assessed by deep transcriptome profiling of both small and messenger RNA from healthy donors’ plasma or serum. Experiments are conducted in triplicate (for a total of 276 transcriptomes) using 189 synthetic spike-in RNAs as processing controls. When comparing blood tubes, so-called blood preservation tubes do not stabilize RNA very well, as is reflected by increasing RNA concentration and number of detected genes over time, and by compromised reproducibility. We also document large differences in RNA purification kit performance in terms of sensitivity, reproducibility, and observed transcriptome complexity. Our results are summarized in 11 performance metrics that enable an informed selection of the most optimal sample processing workflow for your own experiments. In conclusion, we put forward robust quality control metrics for exRNA quantification methods with validated standard operating procedures (SOPs) for processing, representing paramount groundwork for future exRNA-based precision medicine applications.

Custom long non-coding RNA capture enhances detection sensitivity in different human sample types.

Abstract: Long non-coding RNAs (lncRNAs) are a heterogeneous group of transcripts that lack protein coding potential and display regulatory functions in various cellular processes. As a result of their cell- and cancer-specific expression patterns, lncRNAs have emerged as potential diagnostic and therapeutic targets. The accurate characterization of lncRNAs in bulk transcriptome data remains challenging due to their low abundance compared to protein coding genes. To tackle this issue, we describe a unique short-read custom lncRNA capture sequencing approach that relies on a comprehensive set of 565,878 capture probes for 49,372 human lncRNA genes. This custom lncRNA capture approach was evaluated on various sample types ranging from artificial high-quality RNA mixtures to more challenging formalin-fixed paraffin-embedded tissue and biofluid material. The custom enrichment approach allows the detection of a more diverse repertoire of lncRNAs, with better reproducibility and higher coverage compared to classic total RNA-sequencing.

A G316A Polymorphism in the Ornithine Decarboxylase Gene Promoter Modulates MYCN-Driven Childhood Neuroblastoma

Abstract: Ornithine decarboxylase (ODC1), a critical regulatory enzyme in polyamine biosynthesis, is a direct transcriptional target of MYCN, amplification of which is a powerful marker of aggressive neuroblastoma. A single nucleotide polymorphism (SNP), G316A, within the first intron of ODC1, results in genotypes wildtype GG, and variants AG/AA. CRISPR-cas9 technology was used to investigate the effects of AG clones from wildtype MYCN-amplified SK-N-BE(2)-C cells and the effect of the SNP on MYCN binding, and promoter activity was investigated using EMSA and luciferase assays. AG clones exhibited decreased ODC1 expression, growth rates, and histone acetylation and increased sensitivity to ODC1 inhibition. MYCN was a stronger transcriptional regulator of the ODC1 promoter containing the G allele, and preferentially bound the G allele over the A. Two neuroblastoma cohorts were used to investigate the clinical impact of the SNP. In the study cohort, the minor AA genotype was associated with improved survival, while poor prognosis was associated with the GG genotype and AG/GG genotypes in MYCN-amplified and non-amplified patients, respectively. These effects were lost in the GWAS cohort. We have demonstrated that the ODC1 G316A polymorphism has functional significance in neuroblastoma and is subject to allele-specific regulation by the MYCN oncoprotein.

Automatic end-to-end analysis of high-throughput in vitro cell culture screening by HTSplotter

Abstract: Biomedical researchers are moving towards high-throughput screening, as this allows for automatization, better reproducibility and more and faster results. High-throughput screening experiments encompass drug, drug combination, genetic perturbagen or a combination of genetic and chemical perturbagen screens. These experiments are conducted in real-time assays over time or in an endpoint assay. The data analysis consists of data cleaning and structuring, as well as further data processing and visualisation, which, due to the amount of data, can easily become laborious, time consuming, and error-prone. Therefore, several tools have been developed to aid researchers in this data analysis, but they focus on specific experimental set-ups and are unable to process data of several time points and genetic-chemical perturbagen screens together. To meet these needs, we developed HTSplotter, available as web tool and Python module, that performs automatic data analysis and visualisation of either endpoint or real-time assays from different high-throughput screening experiments: drug, drug combination, genetic perturbagen and genetic-chemical perturbagen screens. HTSplotter implements an algorithm based on conditional statements in order to identify experiment type and controls. After appropriate data normalization, HTSplotter executes downstream analyses such as dose-response relationship and drug synergism by the Bliss independence method. All results are exported as a text file and plots are saved in a PDF file. The main advantage of HTSplotter over other available tools is the automatic analysis of genetic-chemical perturbagen screens and real-time assays where results are plotted over time. In conclusion, HTSplotter allows for the automatic end-to-end data processing, analysis and visualisation of various high-throughput in vitro cell culture screens, offering major improvements in terms of versatility, convenience and time over existing tools.

The microRNA Landscape of Acute Beta Cell Destruction in Type 1 Diabetic Recipients of Intraportal Islet Grafts

Abstract: Ongoing beta cell death in type 1 diabetes (T1D) can be detected using biomarkers selectively discharged by dying beta cells into plasma. microRNA-375 (miR-375) ranks among the top biomarkers based on studies in animal models and human islet transplantation. Our objective was to identify additional microRNAs that are co-released with miR-375 proportionate to the amount of beta cell destruction. RT-PCR profiling of 733 microRNAs in a discovery cohort of T1D patients 1 h before/after islet transplantation indicated increased plasma levels of 22 microRNAs. Sub-selection for beta cell selectivity resulted in 15 microRNAs that were subjected to double-blinded multicenter analysis. This led to the identification of eight microRNAs that were consistently increased during early graft destruction: besides miR-375, these included miR-132/204/410/200a/429/125b, microRNAs with known function and enrichment in beta cells. Their potential clinical translation was investigated in a third independent cohort of 46 transplant patients by correlating post-transplant microRNA levels to C-peptide levels 2 months later. Only miR-375 and miR-132 had prognostic potential for graft outcome, and none of the newly identified microRNAs outperformed miR-375 in multiple regression. In conclusion, this study reveals multiple beta cell-enriched microRNAs that are co-released with miR-375 and can be used as complementary biomarkers of beta cell death.

HTSplotter: an end-to-end data processing, analysis and visualisation tool for chemical and genetic in vitro perturbation screening

Abstract: Biomedical researchers are moving towards high-throughput screening, as this allows for automatization, better reproducibility and more and faster results. High-throughput screening experiments encompass drug, drug combination, genetic perturbagen or a combination of genetic and chemical perturbagen screens. These experiments are conducted in real-time assays over time or in an endpoint assay. The data analysis consists of data cleaning and structuring, as well as further data processing and visualisation, which, due to the amount of data, can easily become laborious, time consuming, and error-prone. Therefore, several tools have been developed to aid researchers in this data analysis, but they focus on specific experimental set-ups and are unable to process data of several time points and genetic-chemical perturbagen screens together. To meet these needs, we developed HTSplotter, available as web tool and Python module, that performs automatic data analysis and visualisation of either endpoint or real-time assays from different high-throughput screening experiments: drug, drug combination, genetic perturbagen and genetic-chemical perturbagen screens. HTSplotter implements an algorithm based on conditional statements in order to identify experiment type and controls. After appropriate data normalization, HTSplotter executes downstream analyses such as dose-response relationship and drug synergism by the Bliss independence method. All results are exported as a text file and plots are saved in a PDF file. The main advantage of HTSplotter over other available tools is the automatic analysis of genetic-chemical perturbagen screens and real-time assays where results are plotted over time. In conclusion, HTSplotter allows for the automatic end-to-end data processing, analysis and visualisation of various high-throughput in vitro cell culture screens, offering major improvements in terms of versatility, convenience and time over existing tools.

The MicroRNA Landscape of Acute Beta Cell Destruction in Type 1 Diabetic Recipients of Intraportal Islet Grafts.

Abstract: Ongoing beta cell death in type 1 diabetes (T1D) can be detected using biomarkers selectively discharged by dying beta cells into plasma. microRNA-375 (miR-375) ranks among the top biomarkers based on studies in animal models and human islet transplantation. Our objective was to identify additional microRNAs that are co-released with miR-375 proportionate to the amount of beta cell destruction. RT-PCR profiling of 733 microRNAs in a discovery cohort of T1D patients 1 h before/after islet transplantation indicated increased plasma levels of 22 microRNAs. Sub-selection for beta cell selectivity resulted in 15 microRNAs that were subjected to double-blinded multicenter analysis. This led to the identification of eight microRNAs that were consistently increased during early graft destruction: besides miR-375, these included miR-132/204/410/200a/429/125b, microRNAs with known function and enrichment in beta cells. Their potential clinical translation was investigated in a third independent cohort of 46 transplant patients by correlating post-transplant microRNA levels to C-peptide levels 2 months later. Only miR-375 and miR-132 had prognostic potential for graft outcome, and none of the newly identified microRNAs outperformed miR-375 in multiple regression. In conclusion, this study reveals multiple beta cell-enriched microRNAs that are co-released with miR-375 and can be used as complementary biomarkers of beta cell death.

Recurrent chromosomal imbalances provide selective advantage to human embryonic stem cells under enhanced replicative stress conditions.

Abstract: Human embryonic stem cells (hESCs) and embryonal tumors share a number of common features, including a compromised G1/S checkpoint. Consequently, these rapidly dividing hESCs and cancer cells undergo elevated levels of replicative stress, inducing genomic instability that drives chromosomal imbalances. In this context, it is of interest that long-term in vitro cultured hESCs exhibit a remarkable high incidence of segmental DNA copy number gains, some of which are also highly recurrent in certain malignancies such as 17q gain (17q+). The selective advantage of DNA copy number changes in these cells has been attributed to several underlying processes including enhanced proliferation. We hypothesized that these recurrent chromosomal imbalances become rapidly embedded in the cultured hESCs through a replicative stress driven Darwinian selection process. To this end, we compared the effect of hydroxyurea-induced replicative stress vs normal growth conditions in an equally-mixed cell population of isogenic euploid and 17q + hESCs. We could show that 17q + hESCs rapidly overtook normal hESCs. Our data suggest that recurrent chromosomal segmental gains provide a proliferative advantage to hESCs under increased replicative stress, a process that may also explain the highly recurrent nature of certain imbalances in cancer. This article is protected by copyright. All rights reserved.

Selection and validation of reference genes for accurate RT-qPCR gene expression normalization in cacao beans during fermentation

Abstract: To date, little attention has been paid to the genotypic plasticity and influence of the fermentation process on gene functions and biological processes in cacao beans. The primary tools for such analyses are gene expression studies with reverse transcription quantitative PCR (RT-qPCR). While this is a well-appreciated technique, it is only reliable when considering the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines, which is unfortunately barely applied in plant sciences and non-existent in cacao-related studies. In this study, an appropriate from bean to RT-qPCR protocol was developed. In total, sixty-five candidate reference gene (RG) assays were validated. These assays were either adopted from literature (traditional “housekeeping” genes) or based on RNA-sequencing data (novel). After validation, three novel reference genes (SUGP1, NAP1, SGT1) were recommended for normalization of gene expression within fermented cacao beans. The suitability of the novel candidates surpassed the traditional housekeeping genes. In addition, these assays seemed largely unaffected by RNA integrity. This is the first study to establish a standardized RT-qPCR workflow on cacao beans during fermentation, facilitating future studies. We recommend similar MIQE-based approaches for future gene expression studies on other organisms for miscellaneous objectives.

Exploring the extracellular transcriptome in seminal plasma for non-invasive prostate cancer diagnosis

Abstract: A diagnostic non-invasive biomarker test for prostate cancer at an early stage, with high sensitivity and specificity, would improve diagnostic decision making. Extracellular RNAs present in seminal plasma might contain biomarker potential for the accurate detection of clinically significant prostate cancer. So far, the extracellular messenger RNA (mRNA) profile of seminal plasma has not been interrogated for its biomarker potential in the context of prostate cancer. Here, we investigate the mRNA transcriptome in seminal plasma samples obtained from prostate cancer patients (n=25), patients with benign prostate hyperplasia (n=26) and individuals without prostatic disease (n=6). Seminal plasma harbors a complex mRNA repertoire that reflects prostate as its tissue of origin. The endogenous RNA content is higher in the prostate cancer samples compared to the control samples. Prostate cancer antigen 3 (PCA3), a long non-coding RNA with prostate cancer-specific overexpression, and ATP-binding cassette transporter 1 (ABCA1), known to be involved in the prostate cancer pathogenesis, were more abundant in the prostate cancer group. In addition, twelve high confidence fusion transcripts could be detected in prostate cancer samples, including the bona-fide prostate cancer fusion transcript TMPRSS2-ERG. Our findings provide proof-of-principle that the extracellular transcriptome of seminal plasma can reveal information of an underlying prostate cancer.

Equivalence of saliva RT-qPCR testing to nasal-throat/nasopharyngeal swab testing in the general practitioner's setting to detect SARS-CoV-2

Abstract: Study design Saliva has been proposed as valid alternative for nasopharyngeal swab for RT-qPCR detection of SARS-CoV-2. The sensitivity is generally equivalent, and it comes with much less discomfort for the patient. While there is an overall good performance in the literature for adults, there is much less information on the use of saliva in children or in the general practitioner’s setting. Methods We tested a novel commercially available saliva collection kit with a virus inactivating and RNA stabilizing buffer (InActiv Blue®) in matched saliva and swab samples from 245 individuals, including 216 children, collected by general practitioners. Results Blind RT-qPCR testing of the saliva samples confirmed all 23 positives identified by swab testing (100% concordance), irrespective of age, presence of symptoms, or high-risk status. One child’s saliva sample was found low positive while negative on the nasopharyngeal swab, resulting in an overall relative sensitivity of RT-qPCR saliva testing of 104.3%. Conclusion Saliva collected in InActiv Blue® can be a valid alternative for SARS-CoV-2 RT-qPCR testing in the general practitioner’s setting, including children.

The long non-coding RNA SAMMSON is essential for uveal melanoma cell survival

Abstract: Purpose Long non-coding RNAs (lncRNAs) can exhibit cell-type and cancer-type specific expression profiles, making them highly attractive as therapeutic targets. Pan-cancer RNA sequencing data revealed broad expression of the SAMMSON lncRNA in uveal melanoma (UM), the most common primary intraocular malignancy in adults. Currently, there are no effective treatments for UM patients with metastatic disease, resulting in a median survival time of 6-12 months. We aimed to investigate the therapeutic potential of SAMMSON inhibition in UM. Experimental Design The impact of antisense oligonucleotide (ASO)-mediated SAMMSON inhibition was evaluated in a panel of UM cell lines and patient derived xenograft (PDX) models. Cell proliferation and apoptosis were quantified in vitro and in vivo and complemented with molecular profiles established through RNA-sequencing. SAMMSON interaction partners were identified using ChIRP-MS. Results SAMMSON inhibition impaired the growth and viability of a genetically diverse panel of uveal melanoma cell lines. These effects were accompanied by an induction of apoptosis and were recapitulated in two uveal melanoma PDX models through subcutaneous ASO delivery. SAMMSON pulldown revealed several candidate interaction partners, including various proteins involved in mitochondrial translation. Consequently, inhibition of SAMMSON impaired global protein translation levels and mitochondrial function in uveal melanoma cells. Conclusion SAMMSON expression is essential for uveal melanoma cell survival. ASO-mediated silencing of SAMMSON may provide an effective treatment strategy to treat primary and metastatic uveal melanoma patients.

Custom long non-coding RNA capture enhances detection sensitivity in different human sample types

Abstract: Long non-coding RNAs (lncRNAs) are a heterogeneous group of transcripts that lack protein coding potential and display regulatory functions in various cellular processes. As a result of their cell- and cancer-specific expression patterns, lncRNAs have emerged as potential diagnostic and therapeutic targets. The accurate characterization of lncRNAs in bulk transcriptome data remains challenging due to their low abundance compared to protein coding genes. To tackle this issue, we describe a unique short-read custom lncRNA capture sequencing approach that relies on a comprehensive set of 565,878 capture probes for 49,372 human lncRNA genes. This custom lncRNA capture approach was evaluated on various sample types ranging from artificial high-quality RNA mixtures to more challenging formalin-fixed paraffin-embedded tissue and biofluid material. The custom enrichment approach allows the detection of a more diverse repertoire of lncRNAs, with better reproducibility and higher coverage compared to classic total RNA-sequencing.