Comparison of Fecal MicroRNA Isolation Using Various Total RNA Isolation Kits.

Fecal specimens have long been regarded as promising sources for gastrointestinal cancer screening and have, thus, been extensively investigated in biomarker research. MicroRNAs (miRNAs) are small, non-coding RNA molecules involved in regulating various biological processes. They are commonly dysregulated during tumor development and exhibit differential expression in feces. To assess the preanalytical feasibility of fecal miRNA analysis, we systematically compared the performance of commonly used total RNA extraction methods. Fecal samples from healthy subjects were utilized for this evaluation. Various methods, including miRNeasy, Universal, Trizol, RNeasy, and mirVana kits, were employed to isolate total RNA. MiRNA expression analyses were conducted using TaqMan or SYBR Green qRT-PCR for a subset of miRNAs, with externally spiked-in cel-miR-39 used for normalization. Most methods demonstrated similar performance in terms of the total RNA concentration and purity. Externally spiked cel-miR-39 and endogenous miRNAs (RNU6b, miR-16, and miR-21) exhibited comparable concentrations across the different RNA isolation methods, whereas the RNeasy mini kit consistently yielded lower values. Our findings suggest that various isolation methods produce reproducible and comparable miRNA expression results, supporting the potential comparability and translational applicability of miRNA-based biomarker research in the future.

Real-Time PCR Quantification of 87 miRNAs from Cerebrospinal Fluid: miRNA Dynamics and Association with Extracellular Vesicles after Severe Traumatic Brain Injury.

Severe traumatic brain injury (sTBI) is an intracranial damage triggered by external force, most commonly due to falls and traffic accidents. The initial brain injury can progress into a secondary injury involving numerous pathophysiological processes. The resulting sTBI dynamics makes the treatment challenging and prompts the improved understanding of underlying intracranial processes. Here, we analysed how extracellular microRNAs (miRNAs) are affected by sTBI. We collected thirty-five cerebrospinal fluids (CSF) from five sTBI patients during twelve days (d) after the injury and combined them into d1-2, d3-4, d5-6 and d7-12 CSF pools. After miRNA isolation and cDNA synthesis with added quantification spike-ins, we applied a real-time PCR-array targeting 87 miRNAs. We detected all of the targeted miRNAs, with totals ranging from several nanograms to less than a femtogram, with the highest levels found at d1-2 followed by decreasing levels in later CSF pools. The most abundant miRNAs were miR-451a, miR-16-5p, miR-144-3p, miR-20a-5p, let-7b-5p, miR-15a-5p, and miR-21-5p. After separating CSF by size-exclusion chromatography, most miRNAs were associated with free proteins, while miR-142-3p, miR-204-5p, and miR-223-3p were identified as the cargo of CD81-enriched extracellular vesicles, as characterised by immunodetection and tunable resistive pulse sensing. Our results indicate that miRNAs might be informative about both brain tissue damage and recovery after sTBI.

Construction of an Integrated Device of a Self-Powered Biosensor and Matching Capacitor Based on Graphdiyne and Multiple Signal Amplification: Ultrasensitive Method for MicroRNA Detection.

The detection of microRNA (miRNA) in human serum has great significance for cancer prevention. Herein, a novel self-powered biosensing platform is developed, which effectively integrates an enzymatic biofuel cell (EBFC)-based self-powered biosensor with a matching capacitor for miRNA detection. A catalytic hairpin assembly and hybrid chain reaction are used to improve the analytical performance of EBFC. Furthermore, the matching capacitor is selected as an auxiliary signal amplifying device, and graphdiyne is applied as substrate material for EBFC. The results confirm that the developed method obviously increases the output current of EBFC, and the sensitivity can reach 2.75 µA/pM, which is 786% of pure EBFC. MiRNA can be detected in an expanded linear range of 0.1-100000 fM with a detection limit of 0.034 fM (S/N = 3). It can offer a selective and sensitive platform for nucleotide sequence detection with great potential in clinical diagnostics.

Heterogeneous miRNA-mRNA Regulatory Networks of Visceral and Subcutaneous Adipose Tissue in the Relationship Between Obesity and Renal Clear Cell Carcinoma.

Background: Clear cell renal cell carcinoma (ccRCC) is one of the most lethal urologic cancer. Associations of both visceral adipose tissue (VAT) and subcutaneous adipose tissue (SAT) with ccRCC have been reported, and underlying mechanisms of VAT perhaps distinguished from SAT, considering their different structures and functions. We performed this study to disclose different miRNA-mRNA networks of obesity-related ccRCC in VAT and SAT using datasets from Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA); and find out different RNAs correlated with the prognosis of ccRCC in VAT and SAT. Methods: We screened out different expressed (DE) mRNAs and miRNAs of obesity, in both VAT and SAT from GEO datasets, and constructed miRNA-mRNA networks of obesity-related ccRCC. To evaluate the sensitivity and specificity of RNAs in networks of obesity-related ccRCC in both VAT and SAT, Receiver Operating Characteristic (ROC) analyses were conducted using TCGA datasets. Spearman correlation analyses were then performed to find out RNA pairs with inverse correlations. We also performed Cox regression analyses to estimate the association of all DE RNAs of obesity with the overall survival. Results: 136 and 185 DE mRNAs of obesity in VAT and SAT were found out. Combined with selected DE miRNAs, miRNA-mRNA networks of obesity-related ccRCC were constructed. By performing ROC analyses, RNAs with same trend as shown in networks and statistically significant ORs were selected to be paired. Three pairs were finally remained in Spearman correlation analyses, including hsa-miR-182&ATP2B2, hsa-miR-532&CDH2 in VAT, and hsa-miR-425&TFAP2B in SAT. Multivariable Cox regression analyses showed that several RNAs with statistically significant adjusted HRs remained consistent trends as shown in DE analyses of obesity. Risk score analyses using selected RNAs showed that the overall survival time of patients in the low-risk group was significantly longer than that in the high-risk group regardless of risk score models. Conclusions: We found out different miRNA-mRNA regulatory networks of obesity-related ccRCC for both VAT and SAT; and several DE RNAs of obesity-related ccRCC were found to remain consistent performance in terms of ccRCC prognosis. Our findings could provide valuable evidence on the targeted therapy of obesity-related ccRCC.

Faecal miRNA profiles associated with age, sex, BMI, and lifestyle habits in healthy individuals.

For their stability and detectability faecal microRNAs represent promising molecules with potential clinical interest as non-invasive diagnostic and prognostic biomarkers. However, there is no evidence on how stool miRNA profiles change according to an individual's age, sex, and body mass index (BMI) or how lifestyle habits influence the expression levels of these molecules. We explored the relationship between the stool miRNA levels and common traits (sex, age, BMI, and menopausal status) or lifestyle habits (physical activity, smoking status, coffee, and alcohol consumption) as derived by a self-reported questionnaire, using small RNA-sequencing data of samples from 335 healthy subjects. We detected 151 differentially expressed miRNAs associated with one variable and 52 associated with at least two. Differences in miR-638 levels were associated with age, sex, BMI, and smoking status. The highest number of differentially expressed miRNAs was associated with BMI (n = 92) and smoking status (n = 84), with several miRNAs shared between them. Functional enrichment analyses revealed the involvement of the miRNA target genes in pathways coherent with the analysed variables. Our findings suggest that miRNA profiles in stool may reflect common traits and lifestyle habits and should be considered in relation to disease and association studies based on faecal miRNA expression.

Functional microRNA targetome undergoes degeneration-induced shift in the retina.

BACKGROUND: MicroRNA (miRNA) play a significant role in the pathogenesis of complex neurodegenerative diseases including age-related macular degeneration (AMD), acting as post-transcriptional gene suppressors through their association with argonaute 2 (AGO2) - a key member of the RNA Induced Silencing Complex (RISC). Identifying the retinal miRNA/mRNA interactions in health and disease will provide important insight into the key pathways miRNA regulate in disease pathogenesis and may lead to potential therapeutic targets to mediate retinal degeneration. METHODS: To identify the active miRnome targetome interactions in the healthy and degenerating retina, AGO2 HITS-CLIP was performed using a rodent model of photoreceptor degeneration. Analysis of publicly available single-cell RNA sequencing (scRNAseq) data was performed to identify the cellular location of AGO2 and key members of the microRNA targetome in the retina. AGO2 findings were verified by in situ hybridization (RNA) and immunohistochemistry (protein). RESULTS: Analysis revealed a similar miRnome between healthy and damaged retinas, however, a shift in the active targetome was observed with an enrichment of miRNA involvement in inflammatory pathways. This shift was further demonstrated by a change in the seed binding regions of miR-124-3p, the most abundant retinal AGO2-bound miRNA, and has known roles in regulating retinal inflammation. Additionally, photoreceptor cluster miR-183/96/182 were all among the most highly abundant miRNA bound to AGO2. Following damage, AGO2 expression was localized to the inner retinal layers and more in the OLM than in healthy retinas, indicating a locational miRNA response to retinal damage. CONCLUSIONS: This study provides important insight into the alteration of miRNA regulatory activity that occurs as a response to retinal degeneration and explores the miRNA-mRNA targetome as a consequence of retinal degenerations. Further characterisation of these miRNA/mRNA interactions in the context of the degenerating retina may provide an important insight into the active role these miRNA may play in diseases such as AMD.

PlantMirP2: An Accurate, Fast and Easy-To-Use Program for Plant Pre-miRNA and miRNA Prediction.

MicroRNAs (miRNAs) are a kind of short non-coding ribonucleic acid molecules that can regulate gene expression. The computational identification of plant miRNAs is of great significance to understanding biological functions. In our previous studies, we have put firstly forward and further developed a set of knowledge-based energy features to construct two plant pre-miRNA prediction tools (plantMirP and riceMirP). However, these two tools cannot be used for miRNA prediction from NGS (Next-Generation Sequencing) data. In addition, for further improving the prediction performance and accessibility, plantMirP2 has been developed. Based on the latest dataset, plantMirP2 achieves a promising performance: 0.9968 (Area Under Curve, AUC), 0.9754 (accuracy), 0.9675 (sensitivity) and 0.9876 (specificity). Additionally, the comparisons with other plant pre-miRNA tools show that plantMirP2 performs better. Finally, the webserver and stand-alone version of plantMirP2 are available.

Screening of miRNAs in plasma as a diagnostic biomarker for cardiac disease based on optimization of extraction and qRT-PCR condition assay through amplification efficiency.

BACKGROUND: Although quantitative real-time PCR (qRT-PCR) is a common and sensitive method for miRNAs analysis, it is necessary to optimize conditions and minimize qRT-PCR inhibitors to achieve reliable results. The aim of this study was to minimize interference by contaminants in qRT-PCR, maximize product yields for miRNA analyses, and optimize PCR conditions for the reliable screening of miRNAs in plasma. METHODS: The annealing temperature was first optimized by assessing amplification efficiencies. The effects of extraction conditions on levels of inhibitors that interfere with PCR were evaluated. The tested extraction conditions were the volume of the upper layer taken, number of chloroform extractions, and the inclusion of ethanol washing, a process that reduces PCR interference during RNA extraction using TRIzol. RESULTS: An acceptable amplification efficiency of RT-qPCR was achieved by the optimization of the annealing temperature of the tested miRNAs and by the collection a supernatant volume corresponding to about 50% of the volume of TRIzol with triple chloroform extraction. These optimal extraction and PCR conditions were successfully applied to plasma miRNA screening to detect biomarker candidates for the diagnosis of acute myocardial infarction. CONCLUSION: This is the first study to optimize extraction and qRT-PCR conditions, while improving miRNA yields and minimizing the loss of extracted miRNA by evaluations of the amplification efficiency.

Evaluation of RNA isolation methods for microRNA quantification in a range of clinical biofluids.

BACKGROUND: Extracellular microRNAs (miRNAs), released from cells into biofluids, have emerged as promising biomarkers for diagnostic and prognostic purposes. Several RNA isolation methods are available for the analysis of these cell-free miRNAs by RT-qPCR. Not all methods, however, are equally suitable for different biofluids. Here, we extracted total RNA from four very diverse biofluids: serum, urine, bile, and graft preservation fluid (perfusate). Four different protocols were used: a phenol-chloroform extraction and alcohol precipitation in combination with a precipitation carrier (QP) and three different column-based isolation methods, one with phenol-chloroform extraction (RN) and two without (NG and CU). For this range of clinical biofluid samples, we evaluated the potential of these different RNA isolation methods assessing recovery efficiency and the co-purification of RT-qPCR inhibiting compounds. RESULTS: Differences were observed between each of the RNA isolation methods in the recovery of cel-miR-39, a synthetic miRNA spiked in during the workup procedure, and for endogenous miRNAs. Co-purification of heparin, a known RT-qPCR inhibitor, was assessed using heparinase I during cDNA synthesis. RT-qPCR detection of synthetic miRNAs cel-miR-39, spiked in during RNA workup, cel-miR-54, spiked in during cDNA synthesis, and endogenous miRNAs was strongly improved in the presence of heparinase I for some, but not all, isolation methods. Other, co-isolated RT-qPCR inhibitors were not identified, except for biliverdin, which co-isolated from some bile samples with one of the methods. In addition, we observed that serum and urine contain compounds that enhance the binding of heparin to certain solid-phase columns. CONCLUSIONS: For reliable measurements of miRNA-based biomarkers in biofluids, optimization of RNA isolation procedures is recommended as methods can differ in miRNA detection and in co-purification of RT-qPCR inhibitory compounds. Heparinase I treatment confirmed that heparin appeared to be the major RT-qPCR inhibiting compound, but also biliverdin, co-isolated from bile, could interfere with detection.

Identification and function prediction of novel microRNAs in adenosine monophosphate activated protein kinase-activated Sertoli cells of immature boar.

This study was carried out with the objective to identify function prediction of novel microRNAs (miRNAs) in immature boar Sertoli cells (SCs) treated with 5-aminoimidazole-4-carboxamide-1-ß-D-ribofuranoside (AICAR), which is an agonist of adenosine monophosphate-activated protein kinase (AMPK) for regulating cellular energy homeostasis. Two small RNA libraries (control and AICAR treatment) prepared from immature boar SCs were constructed and sequenced by the Illumina small RNA deep sequencing. We identified 77 novel miRNAs and predicted 177 potential target genes for 26 differential novel miRNAs (four miRNAs up-regulation and 22 miRNAs down-regulation) in AICAR-treated SCs. Gene Ontology enrichment and Kyoto Encyclopedia of Genes and Genomes pathway suggested that target genes of differential novel miRNAs were implicated in many biological processes and metabolic pathways. Our findings provided useful information for the functional regulation of novel miRNAs and target mRNAs on AMPK-activated immature boar SCs.

Isolation of Mitochondria from Liver and Extraction of Total RNA and Protein: Analyses of miRNA and Protein Expression.

Several studies have indicated the presence of microRNAs (miRNAs) within mitochondria although the origin, as well as the biological function, of these mitochondrially located miRNAs is largely unknown. The identification and significance of this subcellular localization is gaining increasing relevance to the pathogenesis of certain disease states. Here, we describe the isolation of highly purified mitochondria from rat liver by differential centrifugation, followed by RNAse A treatment to eliminate contaminating RNA. The coupled extraction of total RNA and protein is a more efficient design for allowing the downstream evaluation of miRNA and protein expression in mitochondria.

Influenza Virus-Induced Novel miRNAs Regulate the STAT Pathway.

MicroRNAs (miRNAs) are essential regulators of gene expression in humans and can control pathogenesis and host-virus interactions. Notably, the role of specific host miRNAs during influenza virus infections are still ill-defined. The central goal of this study was to identify novel miRNAs and their target genes in response to influenza virus infections in airway epithelium. Human airway epithelial cells exposed to influenza A virus (IAV) induced several novel miRNAs that were identified using next-generation sequencing (NGS) and their target genes by biochemical methods. NGS analysis predicted forty-two RNA sequences as possible miRNAs based on computational algorithms. The expression patterns of these putative miRNAs were further confirmed using RT-PCR in human bronchial epithelial cells exposed to H1N1, H9N1(1P10), and H9N1 (1WF10) strains of influenza virus. A time-course study showed significant downregulation of put-miR-34 in H1N1 and put-miR-35 in H9N1(1P10)-infected cells, which is consistent with the NGS data. Additionally, put-miR-34 and put-miR-35 showed a high fold enrichment in an argonaute-immunoprecipitation assay compared to the controls, indicating their ability to form a complex with argonaute protein and RNA-induced silencing complex (RISC), which is a typical mode of action found with miRNAs. Our earlier studies have shown that the replication and survival of influenza virus is modulated by certain transcription factors such as NF-ĸB. To identify the target(s) of these putative miRNAs, we screened 84 transcription factors that have a role in viral pathogenesis. Cells transfected with mimic of the put-miR-34 showed a significant decrease in the expression of Signal Transducers and Activators of Transcription 3 (STAT3), whereas the inhibitor of put-miR-34 showed a significant increase in STAT3 expression and its phosphorylation. In addition, put-miR-34 had 76% homology to the untranslated region of STAT3. NGS and PCR array data submitted to the Gene Ontology project also predicted the role of transcription factors modulated by put-miR-34. Our data suggest that put-miR-34 may be a good target for antiviral therapy.

Evaluation of five column-based isolation kits and their ability to extract miRNA from human milk.

MicroRNA can be found in various body fluids, including breast milk. MicroRNA may be transferred from mother to infant via breast milk and potentially regulate the development of the infant's immune system on a post-transcriptional level. This study aimed to determine the microRNA extraction efficiency of five RNA extraction kits from human skim milk samples. Their efficiency was determined by comparing microRNA concentrations, total RNA yield and purity. Furthermore, hsa-miR-148a-3p expression and the recovery of an exogenous control, cel-miR-39-3p, were quantified using qPCR. Each kit extracted different amounts of microRNA and total RNA, with one kit tending to isolate the highest amount of both RNA species. Based on these results, the extraction kit ReliaPrep™ miRNA Cell and Tissue Miniprep System from Promega was found to be the most appropriate kit for microRNA extraction from human skim milk. Moreover, further research is needed to establish a standardized protocol for microRNA extraction from breast milk.

Comparison of different protocols of RNA preparation from circulating blood for RNA sequencing.

OBJECTIVE: Circulating miRNAs have been extensively used in studies of neurological diseases. Thus, methods to extract high quantity total RNA for RNA sequencing (RNA-seq) and real-time quantitative polymerase chain reaction (RT-qPCR) are needed. However, the extraction of sufficient high-quality nucleic acids from circulating blood is difficult. Differences in eccentricity, cryopreservation conditions and extraction methods may affect RNA quantity and quality. Here, we systematically compared six blood-RNA extraction protocols (protocols 1, 2, 3, 4, 5, and 6; see the methods section for details). RESULTS: Protocol 1 yielded the highest quality and quantity of RNA; protocol 2, protocol 5 and protocol 6 produced RNA of intermediate quality; and protocols 3 and 4 yielded the lowest quality RNA. The RNA integrity number (RIN) for isolated RNA was > 9.0 when protocol 1 or protocol 2 was used, > 8.0 when protocol 5 was used, and > 7.0 when protocol 6 was used; lower values were obtained when protocol 3 or 4 was used. The RNA extracted from circulating blood using protocol 1 was most intact and suitable for RT-qPCR and RNA-seq. CONCLUSIONS: The quality of RNA extracted from circulating blood is affected by high-speed centrifugation and cryopreservation. Adding an RNA stabilizer during the cryopreservation of circulating blood significantly improved RNA quality and quantity. The quality of extracted RNA from circulating blood is improved when using TRIzol relative to that attained with a commercial kit.

Serum microRNA-33 levels in pre-diabetic and diabetic patients.

Diabetes mellitus (DM) is a metabolic disease characterized by hyperglycemia and abnormal insulin secretion. MicroRNAs are small, non-coding RNAs that are able to affect cell biological functions and act as biomarkers for some diseases such as DM. In current study, we measured serum miR-33 in three groups (n = 15) as follows; non-diabetic control, pre-diabetic, and DM patients. Real-time PCR method was used to quantify miR-33 expression. miR-33 expression was significantly increased in pre-diabetic subjects compared to other two groups (p < 0.001). FBS (p < 0.001), insulin (p < 0.001), HOMA-IR (p < 0.001), and TG (p = 0.026) were higher in diabetic subjects than the other two groups. In people that had high physical activity, the number of diabetic subjects were zero and most of them were in pre-diabetic group (p = 0.019). Serum miR-33 level significantly and positively correlated with pre-diabetic state (B = 2.67, p = 0.000), Sex (B = 1.03, p = 0.025), and FBS (B = 0.04, p = 0.036) and also miR-33 was significantly and negatively correlated with HOMA-IR (B = - 1.58, p = 0.04). These findings support the possible role of miR-33 to monitor pre-diabetes onset and progression. It needs to be evaluated in future studies with high number of participants to clarify its mechanism and diagnostic viability.

Expression and Purification of tRNA/ pre-miRNA-Based Recombinant Noncoding RNAs.

Research on RNA function and therapeutic potential is dominated by the use of chemoengineered RNA mimics. Recent efforts have led to the establishment of novel technologies for the production of recombinant or bioengineered RNA molecules, which should better recapitulate the structures, functions and safety profiles of natural RNAs because both are produced and folded in living cells. Herein, we describe a robust approach for reproducible fermentation production of bioengineered RNA agents (BERAs) carrying warhead miRNAs, siRNAs, aptamers, or other forms of small RNAs, based upon an optimal hybrid tRNA/pre-miRNA carrier. Target BERA/sRNAs are readily purified by fast protein liquid chromatography (FPLC) to a high degree of homogeneity (>97%). This approach offers a consistent high-level expression (>30% of total bacterial RNAs) and large-scale production of ready-to-use BERAs (multiple to tens milligrams from 1 L bacterial culture).

Analysis of miRNA Expression Using Digital PCR and the QuantStudio™ 3D Digital PCR System.

miRNAs are short, single-stranded RNA molecules that function as posttranscriptional regulators of gene expression. miRNAs represent ideal biomarkers since they can also circulate in the bloodstream as well as in other biological fluids such as urine, saliva, and cerebrospinal fluid.miRNAs play an important role in the regulation of immune cells including cytotoxic T-lymphocytes. Circulating miRNAs can be analyzed by Real-Time PCR or microarray profiling; however data normalization represents still an unsolved problem due to the lack of widely validated house-keeping miRNAs candidates.Digital PCR (dPCR) is an end-point PCR method that is used for absolute quantification. In this chapter we will describe the applications of Digital PCR for the analysis of miRNAs that can influence immune response in serum samples and we will report a specific protocol that can be used to analyze miRNAs using the QuantStudio™ 3D Digital PCR System. The advantage of this method consists in the possibility to highlight weaker differences in miRNA circulating molecules that can be useful to monitor CTLs behavior in pathological conditions or after therapeutic intervention.

Co-continuous structural effect of size-controlled macro-porous glass membrane on extracellular vesicle collection for the analysis of miRNA.

Recent studies have shown that extracellular vesicles (EVs) can be utilized as appropriate and highly specific biomarkers in liquid biopsy for the diagnosis and prognosis of serious illness. However, there are few methods that can collect and isolate miRNA in EVs simply, quickly and efficiently using general equipment such as a normal centrifuge. In this paper, we developed an advanced glass membrane column (AGC) device incorporating a size-controlled macro-porous glass (MPG) membrane with a co-continuous structure to overcome the limitations of conventional EV collection and miRNA extraction from the EVs. The size of macro-pores in the MPG membrane could be accurately controlled by changing the heating temperature and time on the basis of spinodal decomposition of B2O3, Na2O, and SiO2 in phase separation. The AGC device with an MPG membrane could collect the EVs simply and quickly (< 10 min) from cell culture supernatant, serum and urine. This AGC device could extract miRNA from the EVs captured in the MPG membrane with high efficiency when combined with a miRNA extraction solution. We suggest that the AGC device with an MPG membrane can be useful for the diagnosis and prognosis of serious illness using of EVs in various kinds of body fluids.

Poly-Adenine-Based Spherical Nucleic Acids for Efficient Live-Cell MicroRNA Capture.

Direct delivery of exogenous non-coding nucleic acids into living cells has attracted intense interest in biological applications. However, the cell entry efficiency and target capture ability remain to be improved. Herein, we report a method for compartmenting the nucleic acids on the surface of poly-adenine-based spherical nucleic acids (polyA-SNAs) for efficient capture of oncogenic microRNAs (miRNAs) in living cells. We find that polyA-SNAs exhibit high cell entry efficiency, which is insensitive to the configuration of the anti-miRNA sequences. By programming the length of polyAs, we precisely engineered the spatial configuration of the anti-miRNA sequences in polyA-SNAs. Compartmentalized polyA-SNAs bind to miRNAs with improved capture ability as compared to densely compacted SNAs. We further demonstrate that polyA-SNAs serve as high-efficacy miRNA sponges for capturing oncogenic miRNAs both in living cells and in mice. The efficient inhibition of miRNAs results in significant suppression of tumor growth.

Isolation, Extraction and Deep-Sequencing Analysis of Extracellular RNAs (exRNAs) from Human Plasma.

Extracellular RNAs (exRNAs) are secreted by nearly all cell types and are now known to play multiple physiological roles. Human plasma, a readily available sample for biomedical analysis, was reported to contain various subpopulations of exRNA, some of which are most likely components of plasma ribonucleoproteins (RNPs), while others are encapsulated into extracellular vesicles (EVs) of different size, origin, and composition. Unbiased analysis of exRNA composition can be performed with prefractionation of plasma exRNA followed by library preparation, sequencing, and bioinformatics analysis. In addition to "mature," adaptor ligation-competent RNA species (5'-P/3'-OH), human plasma contains a substantial proportion of degraded RNA fragments, featuring 5'-OH/3'-P or cyclophosphate extremities, which can be made competent for ligation using appropriate treatment. Polyethylene glycol (PEG)-based precipitation kits for EV isolation yield a fraction that is highly contaminated by large RNPs and EV-associated RNAs. Purer EV preparations are obtained by using Proteinase K and RNase A treatment, as well as by size-exclusion chromatography (SEC).