Comprehensive RNA-Sequencing Analysis in Serum and Muscle Reveals Novel Small RNA Signatures with Biomarker Potential for DMD.
Ontology highlight
ABSTRACT:
SUBMITTER: Coenen-Stass AML
PROVIDER: S-EPMC6140421 | biostudies-literature | 2018 Dec
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:We profiled microRNAs (miRNAs) in cell-free serum and plasma samples from human volunteers using deep sequencing of barcoded small RNA cDNA libraries. By introducing calibrator synthetic oligonucleotides during library preparation, we were able to calculate the total as well as specific concentrations of circulating miRNA. Studying trios of samples from newborn babies and their parents we detected placental-specific miRNA in both maternal and newborn circulations and quantitated the relative contribution of placental miRNAs to the circulating pool of miRNAs. Furthermore, sequence variation in the placental miRNA profiles could be traced to the specific placenta of origin. These deep sequencing profiles, which may serve as a model for tumor or disease detection, allow us to define the repertoire of miRNA abundance in the circulation and potential uses as biomarkers.
| S-EPMC3600502 | biostudies-literature
Project description:Extracellular vesicles (EVs) have great potential as a source for clinically relevant biomarkers since they can be readily isolated from biofluids and carry microRNA (miRNA), mRNA, and proteins that can reflect disease status. However, the biological and technical variability of EV content is unknown making comparisons between healthy subjects and patients difficult to interpret. In this study, we sought to establish a laboratory and bioinformatics analysis pipeline to analyse the small RNA content within EVs from patient serum that could serve as biomarkers and to assess the biological and technical variability of EV RNA content in healthy individuals. We sequenced EV small RNA from multiple individuals (biological replicates) and sequenced multiple replicates per individual (technical replicates) using the Illumina Truseq protocol. We observed that the replicates of samples clustered by subject indicating that the biological variability (~95%) was greater than the technical variability (~0.50%). We observed that ~30% of the sequencing reads were miRNAs. We evaluated the technical parameters of sequencing by spiking the EV RNA preparation with a mix of synthetic small RNA and demonstrated a disconnect between input concentration of the spike-in RNA and sequencing read frequencies indicating that bias was introduced during library preparation. To determine whether there are differences between library preparation platforms, we compared the Truseq with the Nextflex protocol that had been designed to reduce bias in library preparation. While both methods were technically robust, the Nextflex protocol reduced the bias and exhibited a linear range across input concentrations of the synthetic spike-ins. Altogether, our results indicate that technical variability is much smaller than biological variability supporting the use of EV small RNAs as potential biomarkers. Our findings also indicate that the choice of library preparation method leads to artificial differences in the datasets generated invalidating the comparability of sequencing data across library preparation platforms.
| S-EPMC6844434 | biostudies-literature
Project description:Urinary stem cells (USCs) are a non-invasive, simple, and affordable cell source to study human diseases. Here we show that USCs are a versatile tool for studying Duchenne muscular dystrophy (DMD), since they are able to address RNA signatures and atypical mutation identification. Gene expression profiling of DMD individuals' USCs revealed a profound deregulation of inflammation, muscle development, and metabolic pathways that mirrors the known transcriptional landscape of DMD muscle and worsens following USCs' myogenic transformation. This pathogenic transcription signature was reverted by an exon-skipping corrective approach, suggesting the utility of USCs in monitoring DMD antisense therapy. The full DMD transcript profile performed in USCs from three undiagnosed DMD individuals addressed three splicing abnormalities, which were decrypted and confirmed as pathogenic variations by whole-genome sequencing (WGS). This combined genomic approach allowed the identification of three atypical and complex DMD mutations due to a deep intronic variation and two large inversions, respectively. All three mutations affect DMD gene splicing and cause a lack of dystrophin protein production, and one of these also generates unique fusion genes and transcripts. Further characterization of USCs using a novel cell-sorting technology (Celector) highlighted cell-type variability and the representation of cell-specific DMD isoforms. Our comprehensive approach to USCs unraveled RNA, DNA, and cell-specific features and demonstrated that USCs are a robust tool for studying and diagnosing DMD.
| S-EPMC8756543 | biostudies-literature
Project description:Angiotensin II (Ang II)-mediated vascular smooth muscle cell dysfunction plays a critical role in cardiovascular diseases. However, the role of microRNAs (miRNAs) in this process is unclear. We used small RNA deep sequencing to profile Ang II-regulated miRNAs in rat vascular smooth muscle cells (VSMC) and evaluated their role in VSMC dysfunction. Sequencing results revealed several Ang II-responsive miRNAs, and bioinformatics analysis showed that their predicted targets can modulate biological processes relevant to cardiovascular diseases. Further studies with the most highly induced miR-132 and miR-212 cluster (miR-132/212) showed time- and dose-dependent up-regulation of miR-132/212 by Ang II through the Ang II Type 1 receptor. We identified phosphatase and tensin homolog (PTEN) as a novel target of miR-132 and demonstrated that miR-132 induces monocyte chemoattractant protein-1 at least in part via PTEN repression in rat VSMC. Moreover, miR-132 overexpression enhanced cyclic AMP-response element-binding protein (CREB) phosphorylation via RASA1 (p120 Ras GTPase-activating protein 1) down-regulation, whereas miR-132 inhibition attenuated Ang II-induced CREB activation. Furthermore, miR-132 up-regulation by Ang II required CREB activation, demonstrating a positive feedback loop. Notably, aortas from Ang II-infused mice displayed similar up-regulation of miR-132/212 and monocyte chemoattractant protein-1, supporting in vivo relevance. In addition, microarray analysis and reverse transcriptase-quantitative PCR validation revealed additional novel miR-132 targets among Ang II-down-regulated genes implicated in cell cycle, motility, and cardiovascular functions. These results suggest that miR132/212 can serve as a novel cellular node to fine-tune and amplify Ang II actions in VSMC.
| S-EPMC3346099 | biostudies-literature
Project description:Maternal diet during gestation and lactation affects the development of skeletal muscles in offspring and determines muscle health in later life. In this paper, we describe the association between maternal low protein diet-induced changes in offspring skeletal muscle and the differential expression (DE) of small non-coding RNAs (sncRNAs). We used a mouse model of maternal protein restriction, where dams were fed either a normal (N, 20%) or a low protein (L, 8%) diet during gestation and newborns were cross-fostered to N or L lactating dams, resulting in the generation of NN, NL and LN offspring groups. Total body and tibialis anterior (TA) weights were decreased in weanling NL male offspring but were not different in the LN group, as compared to NN. However, histological evaluation of TA muscle revealed reduced muscle fibre size in both groups at weaning. Small RNA-sequencing demonstrated DE of multiple miRs, snoRNAs and snRNAs. Bioinformatic analyses of miRs-15a, -34a, -122 and -199a, in combination with known myomiRs, confirmed their implication in key muscle-specific biological processes. This is the first comprehensive report for the DE of sncRNAs in nutrition-associated programming of skeletal muscle development, highlighting the need for further research to unravel the detailed molecular mechanisms.
| S-EPMC8150574 | biostudies-literature
Project description:The circulating microRNA (miRNA) profile has been widely used for identifying potential biomarkers against viral infections. However, data on circulating microRNA expression patterns in dengue patients are scanty. Considering the impact of severity caused by dengue infection, circulating miRNA profiles in plasma of dengue patients may prove to be valuable for developing early prognostic markers for the disease severity. Here, we described an in-depth analytical study of small RNA sequencing data obtained from the plasma of 39 dengue patients. Integrating bioinformatics and in vitro studies, we identified differentially expressed miRNAs (DEMs) (log2 fold change ?1.5, P?<?0.05) associated with dengue disease progression. In comparing miRNA expression pattern with the follow-up samples, nine miRNAs were found to exhibit an altered expression that could distinguish between severe dengue and the convalescent patients. To understand the abundance and specificity of the DEMs in the context of dengue infection and disease progression, eight top-hit DEMs were further validated in the dengue virus-infected cell lines as well as in the patient's plasma and peripheral blood mononuclear cells (PBMCs) using the quantitative reverse transcription-PCR (qRT-PCR) method. Importantly, receiver operating curve analysis further confirmed that the plasma expression pattern of hsa-miR-122-5p could differentiate between different stages of dengue infection (area under the concentration-time curve [AUC]?=?0.792), and dengue-negative patients with other febrile illnesses (AUC?=?0.984). The in silico analysis of DEM target genes suggested an enrichment of the pathways associated with metabolism and inflammation. Our study gives a global view of miRNA expression in the plasma from dengue patients and provides a precious resource of candidate miRNAs involved in dengue infection and disease progression.IMPORTANCE Dengue virus (DENV) infection usually causes dengue fever (DF) with flu-like illness affecting infants, young children, and adults. The DF occasionally evolves into a potentially lethal complication called dengue severe (DS) leading to a rapid fall in platelet count along with plasma leakage, fluid accumulation, respiratory distress, and severe bleeding. The diverse clinical spectrum of dengue disease, as well as its significant similarity to other febrile viral illnesses, makes early identification more challenging in this high-risk group. microRNAs (miRNAs) are small (?19 to 21?nucleotides [nt] in length), noncoding RNAs, extremely stable and easily detectable in the plasma; thus, they have potential as biomarkers for diagnosing and monitoring human diseases. This study provides a comprehensive analysis of miRNAs circulating in plasma of dengue virus-infected patients and identifies the miRNA signatures that have biomarker potential for dengue infection and disease progression.
| S-EPMC7498686 | biostudies-literature
Project description:BackgroundRNA sequencing offers advantages over other quantification methods for microRNA (miRNA), yet numerous biases make reliable quantification challenging. Previous evaluations of these biases have focused on adapter ligation bias with limited evaluation of reverse transcription bias or amplification bias. Furthermore, evaluations of the quantification of isomiRs (miRNA isoforms) or the influence of starting amount on performance have been very limited. No study had yet evaluated the quantification of isomiRs of altered length or compared the consistency of results derived from multiple moderate starting inputs. We therefore evaluated quantifications of miRNA and isomiRs using four library preparation kits, with various starting amounts, as well as quantifications following removal of duplicate reads using unique molecular identifiers (UMIs) to mitigate reverse transcription and amplification biases.ResultsAll methods resulted in false isomiR detection; however, the adapter-free method tested was especially prone to false isomiR detection. We demonstrate that using UMIs improves accuracy and we provide a guide for input amounts to improve consistency.ConclusionsOur data show differences and limitations of current methods, thus raising concerns about the validity of quantification of miRNA and isomiRs across studies. We advocate for the use of UMIs to improve accuracy and reliability of miRNA quantifications.
| S-EPMC6588940 | biostudies-literature
Project description:Background & objectives:Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder and is caused mainly by deletion, duplication and point mutations in the DMD gene. Diagnosis of DMD has been a challenge as the mutations in the. DMD:gene are heterogeneous and require more than one diagnostic strategy for the validation of the mutation. This study was planned to evaluate the targeted next-generation sequencing (NGS) as a single platform to detect all types of mutations in the DMD gene, thereby reducing the time and costs compared to conventional sequential testing and also provide precise genetic information for emerging gene therapies. Methods:The study included 20 unrelated families and 22 patients from an Indian population who were screened for DMD based on phenotypes such as scoliosis, toe walking and loss of ambulation. Peripheral blood DNA was isolated and subjected to multiplex ligation-dependent probe amplification (MLPA) and targeted NGS of the DMD gene to identify the nature of the mutation. Results:In the study patients, 77 per cent of large deletion mutations and 23 per cent single-nucleotide variations (SNVs) were identified. Novel mutations were also identified along with reported deletions, point mutations and partial deletions within the exon of the DMD gene. Interpretation & conclusions:Our findings showed the importance of NGS in the routine diagnostic practice in the identification of DMD mutations over sequential testing. It may be used as a single-point diagnostic strategy irrespective of the mutation type, thereby reducing the turnaround time and cost for multiple diagnostic tests such as MLPA and Sanger sequencing. Though MLPA is a sensitive technique and is the first line of a diagnostic test, the targeted NGS of the DMD gene may have an advantage of having a single diagnostic test. A study on a larger number of patients is needed to highlight NGS as a single, comprehensive platform for the diagnosis of DMD.
| S-EPMC6886143 | biostudies-literature
Project description:The majority of lung cancer deaths are caused by metastatic disease. MicroRNAs (miRNAs) are posttranscriptional regulators of gene expression and miRNA dysregulation can contribute to metastatic progression. Here, small RNA sequencing was used to profile the miRNA and piwi-interacting RNA (piRNA) transcriptomes in relation to lung cancer metastasis. RNA-seq was performed using RNA extracted from formalin-fixed paraffin embedded (FFPE) lung adenocarcinomas (LAC) and brain metastases from 8 patients, and LACs from 8 patients without detectable metastatic disease. Impact on miRNA and piRNA transcriptomes was subtle with 9 miRNAs and 8 piRNAs demonstrating differential expression between metastasizing and non-metastasizing LACs. For piRNAs, decreased expression of piR-57125 was the most significantly associated with distant metastasis. Validation by RT-qPCR in a LAC cohort comprising 52 patients confirmed that decreased expression of miR-30a-3p and increased expression of miR-210-3p were significantly associated with the presence of distant metastases. miR-210-3p tumor cell specificity was evaluated by in situ hybridization and its biomarker potential was confirmed by ROC curve analysis (AUC = 0.839). Lastly, agreement between miRNA-seq and RT-qPCR for FFPE-derived RNA was evaluated and a high level of concordance was determined. In conclusion, this study has identified and validated metastasis-related miRNAs in LAC.
| S-EPMC5432317 | biostudies-literature
Project description:We aimed to characterize the molecular differences and effects from prednisone treatment among IgG4-related disease with salivary gland lesions (RD-SG), without SG lesions (RD-nonSG), and IgG4-related retroperitoneal fibrosis (RF). RNA sequencing was conducted on blood from 25 RD-SG, 11 RD-nonSG, 3 RF and 10 control subjects. Among these, 8 RD-nonSG and 12 RD-SG patients were subjected to treatment with prednisone and/or glucocorticoid-sparing agents. Six RD patients had a longitudinal time point. The mRNA levels of IgG4 and IgE, genes specific for Th2 cells, eosinophils, and neutrophils were over-expressed in RD-SG and RD-nonSG. A B-cell signature was suppressed in patients group versus controls, while Th1, Th2, Treg, and eosinophil gene signatures were increased in patients without treatment. Interestingly, Tfh genes and B cell signature were decreased at flare disease state. Prednisone treatment led to increased neutrophil, but decreased Treg signatures. Serum IgG4 levels correlated with the eosinophil and neutrophil gene signatures in RD-SG patients, and with a B cell signature in only RD-nonSG patients. IgG4, IgE, and cell-specific signatures are regulated in patients, suggesting the imbalance of immune and inflammatory cells in IgG4-related disease. Prednisone treatment selectively modulates Treg, eosinophil, and neutrophil signatures.
| S-EPMC5730556 | biostudies-literature