Detection of circulating extracellular mRNAs by modified small-RNA-sequencing analysis.
Ontology highlight
ABSTRACT:
SUBMITTER: Akat KM
PROVIDER: S-EPMC6538341 | biostudies-literature | 2019 Apr
REPOSITORIES: biostudies-literature
Publications
Detection of circulating extracellular mRNAs by modified small-RNA-sequencing analysis.
Akat Kemal M KM Lee Youngmin A YA Hurley Arlene A Morozov Pavel P Max Klaas Ea KE Brown Miguel M Bogardus Kimberly K Sopeyin Anuoluwapo A Hildner Kai K Diacovo Thomas G TG Neurath Markus F MF Borggrefe Martin M Tuschl Thomas T
JCI insight 20190411
Extracellular mRNAs (ex-mRNAs) potentially supersede extracellular miRNAs (ex-miRNAs) and other RNA classes as biomarkers. We performed conventional small-RNA-sequencing (sRNA-seq) and sRNA-seq with T4 polynucleotide kinase (PNK) end-treatment of total exRNA isolated from serum and platelet-poor EDTA, ACD, and heparin plasma to study the effect on ex-mRNA capture. Compared to conventional sRNA-seq PNK-treatment increased the detection of informative ex-mRNAs reads up to 50-fold. The exRNA pool w ...[more]
Similar Datasets
Project description:Atherosclerosis can occur throughout the arterial vascular system and lead to various diseases. Early diagnosis of atherosclerotic processes and of individual disease patterns would be more likely to be successful if targeted therapies were available. For this, it is important to find reliable biomarkers that are easily accessible and with little inconvenience for patients. There are many cell culture, animal model or tissue studies that found biomarkers at the microRNA (miRNA) and mRNA level describing atherosclerotic processes. However, little is known about their potential as circulating and liquid biopsy markers in patients. In this study, we examined serum-derived miRNA - profiles from 129 patients and 28 volunteers to identify potential biomarkers. The patients had four different atherosclerotic manifestations: abdominal aneurysm (n = 35), coronary heart disease (n = 34), carotid artery stenosis (n = 24) and peripheral arterial disease (n = 36). The samples were processed with an extracellular vesicle enrichment protocol, total-RNA extraction and small RNA-sequencing were performed. A differential expression analysis was performed bioinformatically to find potentially regulated miRNA biomarkers. Resulting miRNA candidates served as a starting point for an overrepresentation analysis in which relevant target mRNAs were identified. The Gene Ontology database revealed relevant biological functions in relation to atherosclerotic processes. In patients, expression of specific miRNAs changed significantly compared to healthy volunteers; 27 differentially expressed miRNAs were identified. We were able to detect a group-specific miRNA fingerprint: miR-122-5p, miR-2110 and miR-483-5p for abdominal aortic aneurysm, miR-370-3p and miR-409-3p for coronary heart disease, miR-335-3p, miR-381-3p, miR493-5p and miR654-3p for carotid artery stenosis, miR-199a-5p, miR-215-5p, miR-3168, miR-582-3p and miR-769-5p for peripheral arterial disease. The results of the study show that some of the identified miRNAs have already been associated with atherosclerosis in previous studies. Overrepresentation analysis on this data detected biological processes that are clearly relevant for atherosclerosis, its development and progression showing the potential of these miRNAs as biomarker candidates. In a next step, the relevance of these findings on the mRNA level is to be investigated and substantiated.
| S-EPMC8546328 | biostudies-literature
Project description:Dysregulation of microRNAs (miRNA) in small extracellular vesicles (sEV) such as exosomes have been implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS). Although circulating cell-free miRNA have been extensively investigated in ALS, sEV-derived miRNAs have not been systemically explored yet. Here, we performed small RNA sequencing analysis of serum sEV and identified 5 differentially expressed miRNA in a discovery cohort of 12 patients and 11 age- and sex-matched healthy controls (fold change > 2, p < 0.05). Two of them (up- and down-regulation of miR-23c and miR192-5p, respectively) were confirmed in a separate validation cohort (18 patients and 15 healthy controls) by droplet digital PCR. Bioinformatic analysis revealed that these two miRNAs interact with distinct sets of target genes and involve biological processes relevant to the pathomechanism of ALS. Our results suggest that circulating sEV from ALS patients have distinct miRNA profiles which may be potentially useful as a biomarker of the disease.
| S-EPMC10073149 | biostudies-literature
Project description:Salivary biomarkers for disease detection, diagnostic and prognostic assessments have become increasingly well established in recent years. In this chapter we explain the current leading technology that has been used to characterize salivary non-coding RNAs (ncRNAs) from the extracellular RNA (exRNA) fraction: HiSeq from Illumina® platform for RNA sequencing. Therefore, the chapter is divided into two main sections regarding the type of the library constructed (small and long ncRNA libraries), from saliva collection, RNA extraction and quantification to cDNA library generation and corresponding QCs. Using these invaluable technical tools, one can identify thousands of ncRNA species in saliva. These methods indicate that salivary exRNA provides an efficient medium for biomarker discovery of oral and systemic diseases.
| S-EPMC5550740 | biostudies-other
Project description:BACKGROUND:Extracellular vesicles (EVs) are nano-sized particles secreted by most cells. EVs carry nucleic acids that hold promise as potential biomarkers in various diseases. Human immunodeficiency virus type 1 (HIV) infects CD4+ T cells and induces immune dysfunction, inflammation, and EV secretion, but little is known about EV small RNA cargo in relation to immune dysregulation in HIV-infected individuals. Here, we characterize small RNA carried by circulating EVs in HIV-positive subjects on antiretroviral therapy (ART) relative to uninfected controls by next-generation RNA sequencing. RESULTS:Plasma EVs isolated from HIV-positive and HIV-negative subjects in test (n?=?24) and validation (n?=?16) cohorts were characterized by electron microscopy, nanoparticle tracking analysis, and immunoblotting for exosome markers. EVs were more abundant in plasma from HIV-positive compared to HIV-negative subjects. Small RNA sequencing of plasma EVs in the test cohort identified diverse small RNA species including miRNA, piRNA, snRNA, snoRNA, tRNA, and rRNA, with miRNA being the most abundant. A total of 351 different miRNAs were detected in plasma EVs, with the top 50 miRNAs accounting for 90% of all miRNA reads. miR-26a-5p was the most abundant miRNA, followed by miR-21-5p and miR-148-3p. qRT-PCR analysis showed that six miRNAs (miR-10a-5p, -?21-5p, -27b-3p, -?122-5p, -146a-5p, -?423-5p) were significantly increased in plasma EVs from HIV-positive compared to HIV-negative subjects in the validation cohort. Furthermore, miR-21-5p, -27b-3p, -146a-5p, and?-?423-5p correlated positively with metabolite markers of oxidative stress and negatively with anti-inflammatory polyunsaturated fatty acids. Over-representation and pathway enrichment analyses of miRNAs and their target genes predicted functional association with oxidative stress responses, interferon gamma signaling, Toll-like receptor signaling, TGF beta signaling, and Notch signaling. CONCLUSIONS:HIV-positive individuals on ART have increased abundance of circulating EVs carrying diverse small RNAs, with miRNAs being the most abundant. Several miRNAs associated with inflammation and oxidative stress are increased in circulating EVs of HIV-positive individuals, representing potential biomarkers of targetable pathways that contribute to disease pathogenesis.
| S-EPMC7656686 | biostudies-literature
Project description:It takes more than 20 years for normal colorectal mucosa to develop into metastatic carcinoma. The long time window provides a golden opportunity for early detection to terminate the malignant progression. Here, we aim to enable liquid biopsy of T1a stage colorectal cancer (CRC) and precancerous advanced adenoma (AA) by profiling circulating small extracellular vesicle (sEV)-derived RNAs. We exhibited a full RNA landscape for the circulating sEVs isolated from 60 participants. A total of 58,333 annotated RNAs were detected from plasma sEVs, among which 1,615 and 888 sEV-RNAs were found differentially expressed in plasma from T1a stage CRC and AA compared to normal controls (NC). Then we further categorized these sEV-RNAs into six modules by a weighted gene coexpression network analysis and constructed a 60-gene t-SNE model consisting of the top 10 RNAs of each module that could well distinguish T1a stage CRC/AA from NC samples. Some sEV-RNAs were also identified as indicators of specific endoscopic and morphological features of different colorectal lesions. The top-ranked biomarkers were further verified by RT-qPCR, proving that these candidate sEV-RNAs successfully identified T1a stage CRC/AA from NC in another cohort of 124 participants. Finally, we adopted different algorithms to improve the performance of RT-qPCR-based models and successfully constructed an optimized classifier with 79.3% specificity and 99.0% sensitivity. In conclusion, circulating sEVs of T1a stage CRC and AA patients have distinct RNA profiles, which successfully enable the detection of both T1a stage CRC and AA via liquid biopsy.
| S-EPMC11315448 | biostudies-literature
Project description:Single cell RNA sequencing (scRNAseq) studies have provided critical insight into the pathogenesis of Severe Acute Respiratory Syndrome CoronaVirus 2 (SARS-CoV-2), the causative agent of COronaVIrus Disease 2019 (COVID-19). scRNAseq workflows are generally designed for the detection and quantification of eukaryotic host mRNAs and not viral RNAs. The performance of different scRNAseq methods to study SARS-CoV-2 RNAs has not been thoroughly evaluated. Here, we compare different scRNAseq methods for their ability to quantify and detect SARS-CoV-2 RNAs with a focus on subgenomic mRNAs (sgmRNAs), which are produced only during active viral replication and not present in viral particles. We present a data processing strategy, single cell CoronaVirus sequencing (scCoVseq), which quantifies reads unambiguously assigned to sgmRNAs or genomic RNA (gRNA). Compared to standard 10X Genomics Chromium Next GEM Single Cell 3' (10X 3') and Chromium Next GEM Single Cell V(D)J (10X 5') sequencing, we find that 10X 5' with an extended R1 sequencing strategy maximizes the unambiguous detection of sgmRNAs by increasing the number of reads spanning leader-sgmRNA junction sites. Differential gene expression testing and KEGG enrichment analysis of infected cells compared with bystander or mock cells showed an enrichment for COVID19-associated genes, supporting the ability of our method to accurately identify infected cells. Our method allows for quantification of coronavirus sgmRNA expression at single-cell resolution, and thereby supports high resolution studies of the dynamics of coronavirus RNA synthesis.ImportanceSingle cell RNA sequencing (scRNAseq) has emerged as a valuable tool to study host-viral interactions particularly in the context of COronaVIrus Disease-2019 (COVID-19). scRNAseq has been developed and optimized for analyzing eukaryotic mRNAs, and the ability of scRNAseq to measure RNAs produced by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has not been fully characterized. Here we compare the performance of different scRNAseq methods to detect and quantify SARS-CoV-2 RNAs and develop an analysis workflow to specifically quantify unambiguous reads derived from SARS-CoV-2 genomic RNA and subgenomic mRNAs. Our work demonstrates the strengths and limitations of scRNAseq to measure SARS-CoV-2 RNA and identifies experimental and analytical approaches that allow for SARS-CoV-2 RNA detection and quantification. These developments will allow for studies of coronavirus RNA biogenesis at single-cell resolution to improve our understanding of viral pathogenesis.
| S-EPMC8629185 | biostudies-literature
Project description:Single-cell RNA sequencing (scRNA-Seq) studies have provided critical insight into the pathogenesis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19). scRNA-Seq library preparation methods and data processing workflows are generally designed for the detection and quantification of eukaryotic host mRNAs and not viral RNAs. Here, we compare different scRNA-Seq library preparation methods for their ability to quantify and detect SARS-CoV-2 RNAs with a focus on subgenomic mRNAs (sgmRNAs). We show that compared to 10X Genomics Chromium Next GEM Single Cell 3' (10X 3') libraries or 10X Genomics Chromium Next GEM Single Cell V(D)J (10X 5') libraries sequenced with standard read configurations, 10X 5' libraries sequenced with an extended length read 1 (R1) that covers both cell barcode and transcript sequence (termed "10X 5' with extended R1") increase the number of unambiguous reads spanning leader-sgmRNA junction sites. We further present a data processing workflow, single-cell coronavirus sequencing (scCoVseq), which quantifies reads unambiguously assigned to viral sgmRNAs or viral genomic RNA (gRNA). We find that combining 10X 5' with extended R1 library preparation/sequencing and scCoVseq data processing maximizes the number of viral UMIs per cell quantified by scRNA-Seq. Corresponding sgmRNA expression levels are highly correlated with expression in matched bulk RNA-Seq data sets quantified with established tools for SARS-CoV-2 analysis. Using this scRNA-Seq approach, we find that SARS-CoV-2 gene expression is highly correlated across individual infected cells, which suggests that the proportion of viral sgmRNAs remains generally consistent throughout infection. Taken together, these results and corresponding data processing workflow enable robust quantification of coronavirus sgmRNA expression at single-cell resolution, thereby supporting high-resolution studies of viral RNA processes in individual cells. IMPORTANCE Single-cell RNA sequencing (scRNA-Seq) has emerged as a valuable tool to study host-virus interactions, especially for coronavirus disease 2019 (COVID-19). Here we compare the performance of different scRNA-Seq library preparation methods and sequencing strategies to detect SARS-CoV-2 RNAs and develop a data processing workflow to quantify unambiguous sequence reads derived from SARS-CoV-2 genomic RNA and subgenomic mRNAs. After establishing a workflow that maximizes the detection of SARS-CoV-2 subgenomic mRNAs, we explore patterns of SARS-CoV-2 gene expression across cells with variable levels of total viral RNA, assess host gene expression differences between infected and bystander cells, and identify non-canonical and lowly abundant SARS-CoV-2 RNAs. The sequencing and data processing strategies developed here can enhance studies of coronavirus RNA biology at single-cell resolution and thereby contribute to our understanding of viral pathogenesis.
| S-EPMC10580996 | biostudies-literature
Project description:BackgroundNorthern blotting is still used as a gold standard for validation of the data obtained from high-throughput whole transcriptome-based methods. However, its disadvantages of lower sensitivity, labor-intensive operation, and higher quality of RNA required limit its utilization in a routine molecular biology laboratory to monitor gene expression at RNA level. Therefore, it is necessary to optimize the traditional Northern protocol to make the technique more applicable for standard use.ResultsIn this paper, we report modifications and tips used to improve the traditional Northern protocol for the detection of mRNAs in total RNA. To maximize the retention of specifically bound radiolabeled probes on the blot, posthybridization washes were performed under only with moderate-stringency until the level of radioactivity retained on the filter decreased to 20~50 counts per second, rather than normally under high and low stringency sequentially for scheduled time or under only high stringent condition. Successful detection of the low-expression gene using heterologous DNA probes in 20 µg of total RNA after a two-day exposure suggested an improvement in detection sensitivity. Quantitatively controlled posthybridization washes combined with an ethidium bromide-prestaining RNA procedure to directly visualize prestained RNA bands at any time during electrophoresis or immediately after electrophoresis, which made the progress of the Northern procedure to be monitored and evaluated step by step, thereby making the experiment reliable and controllable. We also report tips used in the modified Northern protocol, including the moderate concentration of formaldehyde in the gel, the accessory capillary setup, and the staining jar placed into an enamel square tray with a lid used for hybridization. Using our modified Northern protocol, eight rounds of rehybridization could be performed on a single blot. The modification made and tips used ensured the efficient proceeding of the experiment and the resulting good performance, but without using special reagents or equipment.ConclusionsThe modified Northern protocol improved detection sensitivity and made the experiment easy, less expensive, reliable, and controllable, and can be employed in a routine molecular biology laboratory to detect low-expressed mRNAs with heterologous DNA probes in total RNA.
| S-EPMC8772191 | biostudies-literature
Project description:Preeclampsia is one of the most dangerous pregnancy complications, and the leading cause of maternal and perinatal mortality and morbidity. Although the clinical symptoms appear late, its origin is early, and hence detection is feasible already at the first trimester. In the current study, we investigated the abundance of circulating small non-coding RNAs in the plasma of pregnant women in their first trimester, seeking transcripts that best separate the preeclampsia samples from those of healthy pregnant women. To this end, we performed small non-coding RNAs sequencing of 75 preeclampsia and control samples, and identified 25 transcripts that were differentially expressed between preeclampsia and the control groups. Furthermore, we utilized those transcripts and created a pipeline for a supervised classification of preeclampsia. Our pipeline generates a logistic regression model using a 5-fold cross validation on numerous random partitions into training and blind test sets. Using this classification procedure, we achieved an average AUC value of 0.86. These findings suggest the predictive value of circulating small non-coding RNA in the first trimester, warranting further examination, and lay the foundation for producing a novel early non-invasive diagnostic tool for preeclampsia, which could reduce the life-threatening risk for both the mother and fetus.
| S-EPMC5821867 | biostudies-literature
Project description:BACKGROUND: Gene fusion is ubiquitous over the course of evolution. It is expected to increase the diversity and complexity of transcriptomes and proteomes through chimeric sequence segments or altered regulation. However, chimeric mRNAs in pigs remain unclear. Here we identified some chimeric mRNAs in pigs and analyzed the expression of them across individuals and breeds using RNA-sequencing data. RESULTS: The present study identified 669 putative chimeric mRNAs in pigs, of which 251 chimeric candidates were detected in a set of RNA-sequencing data. The 618 candidates had clear trans-splicing sites, 537 of which obeyed the canonical GU-AG splice rule. Only two putative pig chimera variants whose fusion junction was overlapped with that of a known human chimeric mRNA were found. A set of unique chimeric events were considered middle variances in the expression across individuals and breeds, and revealed non-significant variance between sexes. Furthermore, the genomic region of the 5' partner gene shares a similar DNA sequence with that of the 3' partner gene for 458 putative chimeric mRNAs. The 81 of those shared DNA sequences significantly matched the known DNA-binding motifs in the JASPAR CORE database. Four DNA motifs shared in parental genomic regions had significant similarity with known human CTCF binding sites. CONCLUSIONS: The present study provided detailed information on some pig chimeric mRNAs. We proposed a model that trans-acting factors, such as CTCF, induced the spatial organisation of parental genes to the same transcriptional factory so that parental genes were coordinatively transcribed to give birth to chimeric mRNAs.
| S-EPMC3531304 | biostudies-literature