Recent Work & Current Methods for Profiling Extracellular microRNAs

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Webinar – Recent Work & Current Methods for Profiling Extracellular microRNAs In the past, differential profiling and functional studies of microRNA (miRNA) has been performed mainly using tissues samples collected by invasive methods. However, in a clinical setting, more convenient and non-invasive methods are required, such as collection of peripheral blood or other bodily fluids. Recently, it has been demonstrated that miRNAs are present in circulating blood plasma, both free circulating and within exosomes, as well as within other biofluids such as cerebral spinal fluid, urine and even breast milk. These circulating miRNAs represent a potential new approach for diagnostic screening. Webinar Outline • Brief review of microRNA basics: history, biogenesis, function • Recent developments of microRNA research in extracellular fluids and exosomes • Current methods for sample preservation and extraction of various complex extracellular sample types including exosomes • Current methods for microRNA discovery and profiling from extracellular sample types • Case studies and application examples Presented by - Dr. Moemen Abdalla, Norgen Biotek and Dr. Christoph Eicken , LC Sciences

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Recent Work & Current Methods for Profiling Extracellular microRNAs:

Recent Work & Current Methods for Profiling Extracellular microRNAs Dr. Moemen Abdalla Senior Research Scientist Norgen Biotek Corp. moemen@norgenbiotek.com Dr. Christoph Eicken Head of Technical Services – Microarrays LC Sciences, LLC ceicken@lcsciences.com

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Webinar Outline Brief review of microRNA basics: history, biogenesis, function Recent developments of microRNA research in extracellular fluids and exosomes Current methods for sample preservation and extraction of various complex extracellular sample types including exosomes Current methods for microRNA discovery and profiling from extracellular sample types Case studies and application examples

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What is in the Circulation (Bodily Fluids)? Schwarzenbach, H. et al. Nat. Rev. Clin. Oncol. 11, 145–156 (2014 ) [ abstract ]

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MicroRNA MicroRNAs account for approximately 1% of the human genome and are highly conserved in nearly all organisms; In mammals, miRNAs are assumed to regulate more than 50% of all protein-coding genes; There is a growing evidence that miRNAs serve as a ‘ hub ’ in gene regulatory networks that control numerous targets rather than regulate a small number of specific genes.

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MicroRNA Biogenesis Schwarzenbach, H. et al. Nat. Rev. Clin. Oncol. 11, 145–156 (2014 ) [ abstract ]

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Stability of cf-miRNAs in body fluids Data have demonstrated that serum cf-miRNAs remained stable after being subjected to severe conditions that would normally degrade most miRNAs; Despite the presence of RNase activity in plasma, incubation of plasma at room temperature for up to 24 h or being subjeced to up to eight freeze-thaw cycles had minimal effect on cf-miRNA levels; Differential centrifugation and size-exclusion chromatography identified two populations of cf-miRNAs in blood; Protein – bound cf-miRNA cf-miRNA associated with vesicles

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Why Study Extracellular miRNAs? Since miRNA expression is tissue specific, miRNA signature profile has been partially established for each tissue; This specificity together with the remarkable stability of cfmiRNA make miRNAs useful biomarkers with robustness and reproducibility in cancer diagnosis; The assessment of multiple miRNA expression levels, also referred to as miRNA signatures, can accurately predict prognosis in various types of cancers.

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MicroRNA and Cancer MicroRNA functioning as a tumor suppressor

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MicroRNA and Cancer MicroRNA functioning as an Oncogene

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Circulating miRNAs detected in body fluids 2008 Serum - Chen X et al.(2008) Characterization of microRNAs in serum: A novel class of biomarkers for diagnosis of cancer and other diseases. Cell Res 18:997–1006. [ article ] Plasma - Mitchell PS et al.(2008) Circulating microRNAs as stable blood-based markers for cancer detection. PNAS USA 105:10513–10518. [ article ] Exosomes - Taylor DD, Gercel-Taylor C. (2008) MicroRNA signatures of tumor-derived exosomes as diagnostic biomarkers of ovarian cancer. Gynecol Oncol 116(1):153. [ abstract ] Urine - Melkonyan HS et al. (2008) Transrenal nucleic acids: from proof of principle to clinical tests. Ann N Y Acad Sci. 1137:73-81. [ abstract ] Cerebrospinal fluid - Cogswell JP et al. (2008) Identification of miRNA changes in Alzheimer's disease brain and CSF yields putative biomarkers and insights into disease pathways. J Alzheimers Dis 14(1):27-41. [ abstract ] 2009 Saliva - Park NJ et al. (2009) Salivary microRNA: discovery, characterization, and clinical utility for oral cancer detection. Clin Cancer Res 15(17):5473-7. [ article ] 2010 Cows milk - Hata T et al. (2010) Isolation of bovine milk-derived microvesicles carrying mRNAs and microRNAs. Biochem Biophys Res Commun 396(2):528-33. [ abstract ] Breast milk - Kosaka N, Izumi H, Sekine K, Ochiya T. (2010) microRNA as a new immune-regulatory agent in breast milk. Silence. 1(1):7. [ article ] Siddeek et al. 2014

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Diagnostic and prognostic value of circulating miRNAs in patients with different cancer entities Schwarzenbach, H. et al. Nat. Rev. Clin. Oncol. 11, 145–156 (2014 ) [ abstract ]

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Current Challenges in Using miRNAs as Biomarkers Data on circulating miRNAs in some diseases are still limited, and often sample size (number of patients) is small. The concentration of miRNAs in circulating blood is low - Extraction of sufficient amounts can be difficult. Large sample volume may be required. Normalization is tricky – No constitutively expressed circulating miRNAs have been found. No universally implemented guidelines for the collection, extraction, normalization, analysis of samples for miRNA analysis. Various platforms/techniques exist.

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Technical Challenges to analyze miRNAs Levels of cfmiRNA expression are known to be significantly affected by pre-analysis factors, such as; Sample preparation and, RNA extraction methods Different miRNA profiles may be obtained depending on the extraction method which will bias the analysis. Haemolysis is also known to markedly influence miRNA expression patterns, because of a contamination of abundant intracellular miRNAs In other body fluids—such as urine, saliva and pleural effusions— it is also important to remove cells and cell debris completely.

Common RNA Extraction Methods from Bodily Fluids:

Common RNA Extraction Methods from Bodily Fluids Most Bodily Fluids RNA Extraction Products are based on Technologies Developed Historically (circa 1980’s) Phenol:Chloroform Silica Column Chromatography

Phenol:Chloroform Sample Prep has Base-Bias (Loss of Diveristy) :

Phenol:Chloroform Sample Prep has Base-Bias (Loss of Diveristy) Molecular Cell 46, 893–895, June 29, 2012 [ article ] Phenol:Chloroform Extraction Procedures tend to have bias in RNA (particularly miRNA ) recovery based on GC contents

Silica Column Sample Prep has Size-Bias (Loss of Important Small Regulatory RNAs) :

Silica Column Sample Prep has Size-Bias (Loss of Important Small Regulatory RNAs) Total RNA RNA Resolved on Bioanalyzer RNA Resolved on Agarose Gel Electrophoresis Silica Column Total RNA Silica Column Missing Small RNA including microRNA and siRNA

Phenol or Silica Sample Prep was not designed for Bodily Fluids Applications:

Phenol or Silica Sample Prep was not designed for Bodily Fluids Applications Extracelleular RNA Isolation Supplier LT Supplier Q Phenol Use of Phenol Yes Yes Yes Column Chromatography Silica Silica N/A Isolate miRNA Yes Yes Yes Carrier RNA Requirement to achieve Sensitivity Yes Yes Yes Most RNA sample prep products ADAPTS Last Generation Technology to fit into Next Generation Applications

Silicon Carbide as a Matrix for Nucleic Acid & Protein Purification for Next Gen Applications:

Silicon Carbide as a Matrix for Nucleic Acid & Protein Purification for Next Gen Applications Si Si Si Si C Chromatographic Applications: Diversity : Binds All Types of DNA or RNA (including miRNA) without use of Phenol Sensitivity : Superb Sensitivity (Single Cell Level) without use of Carrier Applications : Spin columns, 96-well and Slurry (Liquid Biopsies/Bodily Fluids)

Diversity:

Diversity

True RNA Diversity includes RNA of ALL SIZES, Large and Small:

True RNA Diversity includes RNA of ALL SIZES, Large and Small Norgen Qiagen Norgen Qiagen Small RNA including microRNA and siRNA RNA Resolved on Bioanalyzer RNA Resolved on Agarose Gel Electrophoresis Norgen’s Silicon Carbide Column technology is the only product in the Market that bind RNA sequences at equal rate without bias and without the use of Phenol Norgen’s Total RNA Purification Kit (#17200) Competitor = Qiagen RNeasy Mini Kit

Better miRNA Diversity Recovered = Better Biomarker Discovery from Clinical Samples:

Better miRNA Diversity Recovered = Better Biomarker Discovery from Clinical Samples “We are amazed not only by the total amount of miRNA but also by how many different miRNAs can be detected. In fact, this is way more than you use in an average tissue sample.” (Customer Testimonial, Based on LC Sciences miRNA ) Norgen’s Total RNA Purification Kit (#17200) Competitor = Ambion mirVana Kit Norgen (100 m L Plasma) mirVana (600 m L Plasma)

True RNA Diversity - Consistent and Un-Biased:

True RNA Diversity - Consistent and Un-Biased microRNA mRNA Norgen Trizol miRNeasy Trizol has high variation in miRNA recovery  “Nucleating Effect” of Alcohol Precipitation Phenol + Silica-based Total RNA Kit ( miRNeasy ) has high variation in mRNA recovery  Extremely High Alcohol Concentration to Bind RNA to Column (“Precipitate-Out Effect”) Norgen’s Total RNA Purification Kit (#17200) Competitor = Trizol and Qiagen miRNeasy Kit RNA Isolation from Cultured Cell p53 RT-PCR Panel

Sensitivity:

Sensitivity

Emerging and Challenging Biological Sample Requiring Both Diversity and Sensitivity (Exosomes):

Emerging and Challenging Biological Sample Requiring Both Diversity and Sensitivity (Exosomes) Eldh et al ( Uof Gothenburg). 2012. Molecular Immunology 50:278-286 RNA Isolated from Exosome (Ultracentrifugation ) [ abstract ] Norgen’s Total RNA Purification Kit (#17200, Identical to miRCURY RNA Kit) Competitor = Ambion mirVana , Qiagen miRNeasy , Trizol Norgen Norgen Cellular RNA Exosome RNA Best Yield and Best RIN Value Best Yield and Best Size Diversity of RNA

Recovering Both Large and Small RNA in Emerging Biological Sample (Urine Exosomes):

Recovering Both Large and Small RNA in Emerging Biological Sample (Urine Exosomes) Urine Exosome RNA – Ion PGM Cheng et al 2013 (U of Melbourne). Kidney International. Norgen’s Urine Exosome RNA Kit (#47200) 1) RNA Diversity – Large and Small 2) Scalable input 3) Ease of Use – Shortest Time

Recovering Best RNA Diversity in Emerging Biological Sample (Plasma cfmiRNA):

Recovering Best RNA Diversity in Emerging Biological Sample (Plasma cfmiRNA) Plasma Exosome RNA – LC Sciences microRNA Microarray Norgen’s Plasma/Serum RNA Kit (#55000) Compared to Ambion mirVana Kit, Qiagen miRNeasy and Exiqon Kit for Biofluids 1) RNA Diversity – More miRNA recovered 2) Ease of Use – Shortest Time

Ease of Use:

Ease of Use

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Phase Separation 10 minutes RNA Precipitation 10 minutes RNA Pellet Wash 5 minutes Lysate Preparation 5 minutes Phenol ( Trizol ) 30+ mins Total RNA ~15 mins RNA without miRNA Silica ( RNeasy ) Bind RNA 1 minute Wash 3 x 1 min. Elute RNA 3 minute Lysate Preparation 5 minutes SiC (Norgen) Bind RNA 1 minute Wash 3 x 1 min. Elute RNA 3 minute Lysate Preparation 5 minutes ~15 mins True Total RNA Silica ( miRNeasy ) Lysate Preparation 5 minutes Phase Separation 10 minutes Wash 3 x 1 min. Elute RNA 1 minute Bind RNA 1 minute ~25 mins Total RNA Extraction Methods for cf-RNA Diversity

Better miRNA Diversity Recovered = Better Biomarker Discovery from Clinical Samples:

Better miRNA Diversity Recovered = Better Biomarker Discovery from Clinical Samples Total RNA Purification Kit (#17200) Affymetrix microRNA microarrays with 400 uL Plasma Godfrey et al (NIH) Breast Cancer Research 2013, 15:R42 [ article ] Large Scale NIH Breast Cancer Biomarker Screening involving Serum obtained from 410 individuals Trizol or Phenol-Silica Kit: An equivalent study will require 410 Phenol:Chloroform Extractions

Issues Associated with miRNA Measurement (Quantity and Quality):

Issues Associated with miRNA Measurement (Quantity and Quality) One of the main problems associated with circulating miRNA extraction and comparison of sample collectives is the quantification of the miRNA; Measuring the amount of cf-RNA isolated from body fluid samples is especially difficult due to the low concentration of RNA typically obtained from these samples; The low abundance of miRNA in bodily fluids can hardly be determined using spectrophotometers, nanodrop, bioanalyzer, etc... Only relative quantification by a stem-loop reverse transcription qPCR (RT-qPCR)

Bodily Fluids cf-RNA – What to Expect?:

Bodily Fluids cf-RNA – What to Expect? The amount of cell-free RNA significantly varies between sample – to – sample. Normally, RNA yield from bodily fluids is highly variable Very Low RNA Concentration = Low A260/280 No rRNA (28S and 18S) on Bioanalyzer or 1-1.5% agarose gel Low RIN Values

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microRNA Research Tools Detection & Profiling Northern Blotting In situ hybridization Real-time PCR Microarray analysis Next-gen Sequencing Target Determination Bioinformatics – TargetScan , PicTar , PITA Gene / proteome expression analysis Pull-down assays 5′ RACE analyses Degradome Sequencing Functional Analysis Lucifierase Assays Gene knockout/overexpression models miRNA inhibition - antagomirs miRNA mimicry miRNA Identification Genetic screening Direct cloning, sequencing Computational strategy – MIRCheck , findMiRNA , MIRscan , MiRAlign Tiling Microarrays Next-gen Sequencing Pathway Analysis Bioinformatics – miRFocus , mirPath , MMIA Databases miRandola HMDD v2.0 Seq-Array SM ACGT101-miR Stem-Loop Specific Degradsome Seq Digital Gene Expression Pathway Network

miRandola: extracellular circulating microRNAs database:

miRandola: extracellular circulating microRNAs database Russo F, Di Bella S, Nigita G, Macca V, Laganà A, Giugno R, Pulvirenti A, Ferro A. (2012) miRandola : extracellular circulating microRNAs database . PLoS One 7(10):e47786. [ article ] Comprehensive classification of different extracellular circulating miRNA types. Direct link to the knowledge base miRò & data mining facilities (updated in 2014 to include) Systematic comparison of expression profiles of cellular and extracellular miRNAs miRNA targets enrichment analysis procedure Information on drugs and their effect on miRNA expression Allow users to infer possible biological functions of the circulating miRNAs and their connection with the phenotype.

MicroRNA Profiling Review::

MicroRNA Profiling Review: MicroRNA profiling: approaches and considerations Colin C. Pritchard, Heather H. Cheng & Muneesh Tewari Nature Reviews Genetics 13, 358-369 (May 2012) .[ abstract ]

Microarray vs RNA Sequencing:

Microarray vs RNA Sequencing Key Advantages of Microarray Robust, reliable method, proven over decades of use High through-put method – 100s of samples analyzed per month Streamlined handling – can be easily automated Straightforward data analysis Short turn-around time – 5 days Lower cost Key Advantages of RNA- Seq Provides a comprehensive view of the transcriptome . All transcripts can be analyzed (mRNA, ncRNA , snoRNA , lncRNA , miRNA , ...). Not necessarily dependent on any prior sequence knowledge. Increased dynamic range and tunable sensitivity. Can detect structural variations such as gene fusions and alternative splicing events. A truly digital solution

microRNA Microarray Expression Profiling:

microRNA Microarray Expression Profiling Comprehensive Microarray Experiment Sample QC Sample preparation Hybridization reactions Data extraction, processing & analysis GEO submission In sync with miRBase (Version 21) Customer Total RNA Small RNA Isolation Labeling Customer Sequences Chip Design Chip Synthesis Chip QC Hybridization Signal Amplification Image Acquisition Customer Analysis Report Data Extraction Data Analysis Sample QC miRBase

microRNA Microarray Expression Profiling:

microRNA Microarray Expression Profiling Control Treated Biological repeats t-Test p < 0.05 p < 0.01 Control Treated Multi-array normalization and clustering analysis Array assay Differentiated miRNAs of Biological & Statistical Significance - Multiple Chips

Microarray vs RNA Sequencing:

Microarray vs RNA Sequencing Key Advantages of Microarray Robust, reliable method, proven over decades of use High through-put method – 100s of samples analyzed per month Streamlined handling – can be easily automated Straightforward data analysis Short turn-around time – 5 days Lower cost Key Advantages of RNA-Seq Provides a comprehensive view of the transcriptome. All transcripts can be analyzed (mRNA, ncRNA, snoRNA, lncRNA, miRNA, ...). Not necessarily dependent on any prior sequence knowledge. Increased dynamic range and tunable sensitivity. Can detect structural variations such as gene fusions and alternative splicing events. A truly digital solution

Small RNA Sequencing and Data Analysis:

Small RNA Sequencing and Data Analysis Comprehensive RNA Sequencing Experiment Sample QC Sample preparation Library preparation High-throughput sequencing Advanced bioinformatics analysis High level customer support

Reg. Experimental Design Sample Replicates for Expression Studies:

Reg. Experimental Design Sample Replicates for Expression Studies Biological Replicates – Still Very Important For experiments performed with a small number of biological replicates, significant results may be due to biological diversity between individuals and may not be reproducible - it is impossible to know whether expression patterns are specific to the individuals in the study or are a characteristic of the test condition. There is no statistical significance for a difference observed between 2 samples. There is no magic to RNA-Seq. These ideas are widely accepted for DNA microarray experiments, where a large number of biological replicates are now required to justify scientific conclusions. Hansen KD, Wu Z, Irizarry RA, Leek JT.  2011 Sequencing technology does not eliminate biological variability.  Nat Biotechnol  29:572–573. [ abstract ]

Small RNA Sequencing and Data Analysis:

Small RNA Sequencing and Data Analysis Instrument: Illumina HiSeq 2500 Length of Reads: 50 bases Number of Reads: ~100 Million Data Output: ~10-10000 Gb Bar-coding (Indexing) Samples: We recommend 10 samples per lane The total number of reads does not change with bar-coding Sacrifice sequencing depth for lower cost   Total Reads / Lane Number of Samples / Lane Reads/ Sample 100 M 1 100 M 100 M 2 50 M 100 M 3 33 M 100 M 4 25 M 100 M 5 20 M 100 M 6 16 M

Small RNA Sequencing and Data Analysis:

Small RNA Sequencing and Data Analysis Data Flow Mappable reads Raw reads Reads mapped to mammalian mirs in miRBase Reads un-mapped to mammalian mirs in miRBase mirs mapped to species genome mirs un-mapped to species genome Reads mapped to species genome Reads un-mapped to species genome Reads un-mapped to mRNA, Rfam, and repbase Reads mapped to mRNA, Rfam, and repbase Reads mapped to species genome Reads un-mapped to species genome Group 4 no hit others Group 1 Group 2 Group 3 Known species miRNAs Known miRNAs candidate species miRNAs Candidate species miRNAs genome inconsistent with miRBase Potentially novel miRNAs ACGT101-miR v3.5 Software

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Urinary Exosomes Urinary Exosome miRNome Analysis and its Applications to Salt Sensitivity of Blood Pressure Gildea JJ, Carlson JM, Schoeffel CD, Carey RM, Felder RA. (2013) Urinary Exosome miRNome Analysis and its Applications to Salt Sensitivity of Blood Pressure . Clin Biochem [Epub ahead of print]. [ abstract ] Investigating renal cellular pathophysiology through the study of kidney cell-derived extracellular biomarkers has the potential of providing a deeper understanding of how individuals express unique patterns of salt sensitivity of blood pressure. Salt sensitivity of blood pressure (BP) is associated with higher incidence of cardiovascular disease independent of hypertension. Human urinary exosomal miRNome was examined by microarray. Of 1898 probes tested, the expression of 45 urinary exosomal miRNAs associates with an individual's blood pressure response to sodium. Differential miRNA expression in urine exosomes . In the heat maps above, each column corresponds to the relative expression profile of a pooled urine sample from an individual previously indexed for their blood pressure response to salt. The bar graph on the right displays the absolute RFU of each specific miRNA probe.

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Breast Milk Exosomes Lactation-Related MicroRNA Expression Profiles of Porcine Breast Milk Exosomes Gu Y, Li M, Wang T, Liang Y, Zhong Z, Wang X, Zhou Q, Chen L, Lang Q, He Z, Chen X, Gong J, Gao X, Li X, Lv X. (2012) Lactation-Related MicroRNA Expression Profiles of Porcine Breast Milk Exosomes . PLoS ONE 7(8): e43691. [ article ] Analyzed miRNA expression profiles in porcine milk exosomes across the entire lactation period by deep sequencing. Found that immune-related miRNAs are present and enriched in breast milk exosomes (p<10−16, χ2 test) and are generally resistant to relatively harsh conditions. Notably, these exosomal miRNAs are present in higher numbers in the colostrums than in mature milk. It was higher in the serum of colostrum-only fed piglets compared with the mature milk-only fed piglets. These immune-related miRNA-loaded exosomes in breast milk may be transferred into the infant body via the digestive tract. Thirteen well-characterized immune-related miRNAs. The data are normally distributed (Kolmogorov-Smirnov test, p>0.05). The statistical significance was calculated by Student’s t-test (n = 6 per group). Values are means±SD. CF: colostrum-only feeding; MF: mature milk-only feeding.

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Extracellular vesicles Characterization of the small RNA content of Trypanosoma cruzi extracellular vesicles Bayer-Santos E, Lima Fb , Ruiz JC, Almeida IC, d Si. (2014) Characterization of the small RNA content of Trypanosoma cruzi extracellular vesicles. Mol Biochem Parasitol [Epub ahead of print]. [ article ] Trypanosoma cruzi is a parasite that causes Chagas disease in humans and other animals. Performed a deep sequencing and genome-wide analysis of Trypanosoma cruzi -derived extracellular vesicles secreted by noninfective epimastigote and infective metacyclic trypomastigote forms. Data show that the small RNAs contained in these extracellular vesicles originate from multiple sources, including tRNAs . In addition, these results reveal that the variety and expression of small RNAs are different between parasite stages, suggesting diverse functions. Taken together, these observations call attention to the potential regulatory functions that these RNAs might play once transferred between parasites and/or to mammalian host cells.

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Serum/Plasma Parasite-derived Circulating miRNAs are Found in Plasma or Serum Even for Species that Do Not Live in the Bloodstream Tritten L, Burkman E, Moorhead A, Satti M, Geary J Detection of Circulating Parasite-Derived MicroRNAs in Filarial Infections. (2014) PLoS Negl Trop Dis 8(7): e2971. doi:10.1371/journal.pntd.0002971 [ article ] Filarial parasites commonly infect humans and animals, especially in tropical settings, and cause debilitating disease. Investigated the potential of parasite-derived microRNAs as biomarkers of infection. Using deep-sequencing technologies and bioinformatics.   Identified over two-hundred mature miRNA candidates of nematode origin in plasma from Dirofilaria immitis -infected dogs. Discovered twenty-one miRNA candidates predicted to be released by Onchocerca volvulus in infected human sera. Developed two RT-qPCR assays for the detection of D. immitis miR-71 and miR-34 in dog plasma that discriminated infected from uninfected samples.

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Serum/Plasma A Pilot Study of Serum MicroRNAs Panel as Potential Biomarkers for Diagnosis of Nonalcoholic Fatty Liver Disease Tan Y, Ge G, Pan T, Wen D, Gan J. (2014) A Pilot Study of Serum MicroRNAs Panel as Potential Biomarkers for Diagnosis of Nonalcoholic Fatty Liver Disease. PLoS ONE 9(8), e105192. [ article ] Serum miRNA expression was investigated (by Illumina sequencing) using three cohorts comprising 465 participants (healthy controls and NAFLD patients). Identified an miRNA panel (hsa-miR-122-5p, hsa-miR-1290, hsa-miR-27b-3p, and hsa-miR-192-5p) with a high diagnostic accuracy for NAFLD (sensitivity = 90.3%, specificity = 76.2%). The results indicate that the miRNA panel is a more sensitive and specific biomarker for NAFLD than ALT (Alanine Aminotransferase) and FIB-4 (Fibrosis-4). Relative expression of miRNAs between controls and NAFLD patients.

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miRNA P value Group 1 Group 2 Group 3 NGT Pre-diabetes (IGT/IFG) T2D Nominal CT mean Nominal CT SD Nominal CT mean Nominal CT SD Nominal CT mean Nominal CT SD hsa-let-7i 1.53E−02 25.09 1.05 25.46 1.00 25.99 0.98 hsa-miR-23a 2.85E−05 24.86 0.98 25.49 0.85 26.11 0.66 hsa-miR-486 6.62E−02 21.22 1.25 21.33 1.16 21.99 1.12 hsa-miR-186 3.20E−02 28.59 0.97 28.62 1.01 29.27 0.89 hsa-miR-191 3.32E−02 25.05 0.71 25.03 0.98 25.63 0.85 hsa-miR-192 2.31E−02 30.01 1.50 30.16 1.30 30.98 0.91 hsa-miR-96 3.36E−02 31.81 1.51 32.44 1.35 32.95 1.36 hsa-miR-199a 4.41E−01 28.72 0.75 28.86 0.88 29.05 0.87 hsa-miR-122 2.28E−01 27.10 2.93 27.52 2.02 28.22 1.42 hsa-miR-146a 1.02E−01 28.31 0.76 28.49 0.74 28.79 Serum/Plasma Serum miR-23a, a potential biomarker for diagnosis of pre-diabetes and type 2 diabetes Yang Z, Chen H, Si H, Li X, Ding X, Sheng Q, Chen P, Zhang H. (2014) Serum miR-23a, a potential biomarker for diagnosis of pre-diabetes and type 2 diabetes. Acta Diabetol [Epub ahead of print]. [ abstract ] miRNAs regulate several metabolic pathways including insulin secretion, glucose homeostasis. Explored serum miRNA profiles (next-gen sequencing followed by qRT -PCR) in T2D patients to search for candidate biomarker miRNAs . The levels of 8 miRNAs demonstrated a significant decline in T2D patients compared with pre-diabetes patients. These results also revealed that serum miR-23a was a valuable biomarker for early detection of T2D and pre-diabetes with NGT. The sensitivity for this marker was 79.2 % and the specificity was 75.0 % The levels of eight miRNAs were shown to be significantly decreased in T2D patients relative to normal NGT controls. And only miR-23a, was expressed significantly different between pre-diabetes patients to normal NGT controls

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Serum/Plasma Differentially expressed miRNAs in the plasma may provide a molecular signature for aggressive pancreatic cancer Ali S, Almhanna K, Chen W, Philip PA, Sarkar FH. (2010) Differentially expressed miRNAs in the plasma may provide a molecular signature for aggressive pancreatic cancer. Am J Transl Res 3(1), 28-47. [ article ] Pancreatic cancer (PC) has the poorest overall survival rate among all human cancers because of late diagnosis and absence of screening tools. Used microarray to compared the expression profile of miRNAs in the plasma of patients diagnosed with PC with healthy volunteers. Expression of miR-21 was correlated with worse survival Expression of let-7 was inversely correlated with survival miR-21 family was markedly over-expressed in chemo-resistant PC cell lines Results suggest that expression of miRNAs in newly diagnosed patients could serve as potential biomarker for tumor aggressiveness, and such miRNAs could be useful for the screening of high-risk patients. miRNA expression and survival (A) Box plot representing the expression of seven miRNAs as assessed by qRT-PCR. (B) Kaplan-Meier curves and log-rank tests for miR-21 expression and patient survival. (C) Kaplan-Meier curves and log-rank tests for let-7d expression and patient survival. These results suggest that higher expression of miR-21 could serve as a biomarker for worse survival of PC patients, and thus could serves as an important prognostic marker.

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