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Steps to achieve quantitative measurements of microRNA using two-step droplet digital PCR
Droplet digital PCR (ddPCR) is being advocated as a reference method to measure rare genomic targets. It has consistently been proven to be more sensitive and direct at discerning copy numbers of DNA than other quantitative methods. However, one of the largest obstacles to measuring microRNA (miRNA) using ddPCR is that reverse transcription efficiency depends upon the target, meaning small RNA nucleotide composition directly effects primer specificity in a manner that prevents traditional quantitation optimization strategies. Additionally, the use of reagents that are optimized for miRNA measurements using quantitative real time PCR (qRT PCR) appear to either cause false positive or false negative detection of certain targets when used with traditional ddPCR quantification methods. False readings are often related to using inadequate enzymes, primers and probes. Given that two step miRNA quantification using ddPCR relies solely on reverse transcription and uses proprietary reagents previously optimized only for qRT PCR, these barriers are substantial. Therefore, here we outline essential controls, optimization techniques, and an efficacy model to improve the quality of ddPCR miRNA measurements. We have applied two step principles used for miRNA qRT PCR measurements and leveraged the use of synthetic miRNA targets to evaluate ddPCR following cDNA synthesis with four different commercial kits. We have identified inefficiencies and limitations as well as proposed ways to circumvent identified obstacles. Lastly, we show that we can apply these criteria to a model system to confidently quantify miRNA copy number. Our measurement technique is a novel way to quantify specific miRNA copy number in a single sample, without using standard curves for individual experiments. Our methodology can be used for validation and control measurements, as well as a diagnostic technique that allows scientists, technicians, clinicians, and regulators to base miRNA measures on a single unit of measurement rather than a ratio of values.
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Quantitative Metagenomics Benchmarking Experiment Data Set
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To assess the variability of low-abundance oligonucleotide detection across sample matrices, we spiked DNA reference standards (meta sequins) into replicate wastewater DNA extracts at logarithmically decreasing mass-to-mass percentages (m/m%) and deeply sequenced them on the Illumina platform. This dataset summarizes the experimental conditions and results of the detection frequencies of those oligonucleotides as well as detailed descriptions of the DNA reference standards used. This dataset is associated with the following publication: Davis, B., P. Vikesland, and A. Pruden. Evaluating Quantitative Metagenomics for Environmental Monitoring of Antibiotic Resistance and Establishing Detection Limits. ENVIRONMENTAL SCIENCE & TECHNOLOGY. American Chemical Society, Washington, DC, USA, 59(12): 6192-6202, (2025).
Mining microarray expression data by literature profiling
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The lack of efficient techniques for assessing the biological implications of microarray gene-expression data remains an important obstacle in exploiting this information. To address this need, a mining technique has been developed based on the analysis of literature profiles generated by extracting the frequencies of certain terms from thousands of abstracts stored in the Medline literature database.
Differential expressed mRNA and microRNA from expression profiling by RNA and small RNA sequencing
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data was from HepG2 cells treated with nano-silver particles using silver nitrate as negative controls. Differentially expressed messenger RNA and microRNA were obtained by RNA sequencing and data analysis. Differentially expressed RNA and microRNA lists were than uploaded to Ingenuity Pathway Analysis to find the pathways altered by the differentially expressed genes. This dataset is associated with the following publication: Thai, S., C. Jones, B. Robinette, H. Ren, B. Vallanat, A. Fisher, and K. Kitchin. Effects of Silver Nanoparticles and Silver Nitrate on mRNA and microRNA Expression in Human Hepatocellular Carcinoma Cells (HepG2). Journal of Nanoscience and Nanotechnology. American Scientific Publishers, VALENCIA, CA, USA, 21(11): 5414-5428, (2021).
Measuring microRNAs in LCM-isolated nephron segments to determine region-specific expression
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To determine nephron specificity of microRNA expression that may be associated as toxicity biomarker seen in the urine, laser-capture microdissection (LCM) was performed on the 4 major segment of the nephron in control rat samples. These samples were sent to Bayer for candidate digital drop PCR measurements, NIEHS for smallRNA-seq measurements, and Abcam for FirePlex measurements. smallRNA-seq measurements were used as global assessment of miRNA expression in these segments. FirePlex and ddPCR were used as medium to low number candidate assessments, respectively, of miRNA candidates, based on seq and urine data. "Data associated with Fig 4" summarizes the ddPCR findings. "Data Associated with Figure 5" and "Data Associated with Figure 6" summarize the smallRNA-seq findings. "Data Associated with Figure 7" summarize the FirePlex findings. "Data associated with Figure 8_9" summarize the correlations of these measurements. The cumlmination of these data determine nephron-specific and enriched expression of miRNAs, that are potential candidates of toxicity markers that could leak into the urine upon chemical exposure. This dataset is associated with the following publication: Chorley, B., H. Ellinger-Ziegelbauer, M. Tackett, F. Simutis, A. Harrill, J. McDuffie, E. Atabakhsh, R. Nassirpour, L. Whiteley, J. Léonard, G. Carswell, E. Harpur, C. Chen, and J. Gautier. Urinary miRNA Biomarkers of Drug-Induced Kidney Injury and Their Site Specificity Within the Nephron. TOXICOLOGICAL SCIENCES. Society of Toxicology, RESTON, VA, 180(1): 1-16, (2021).
Dataset for Figure 1 - Leveling trend of published microRNA biomarker studies in common biofluids.
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PubMed was searched for the terms microRNA and biomarker in addition to blood (blue), urine (orange), saliva (grey), or cerebrospinal fluid (yellow). The graph in the paper displays the number of total annual publications over the past 16 years and the actual numbers used generate this graph are in incuded in the attached spreadsheet. A near annual doubling of publications occurred from the years 2009 until 2015, whereas only a mere 4% increase in annual publications from 2015 until 2018 was noted. While a number of factors can contribute to these publication trends, this indicates an overall cooling of research interest in biofluid-based microRNA biomarker development. This dataset is associated with the following publication: Chorley, B., E. Atabakhsh, G. Doran, J. Gautier, H. Ellinger-Ziegelbauer, D. Jackson, T. Sharapova, P. Yuen, R. Church, P. Couttet, R. Froetschl, J. McDuffie, V. Martine, P. Pande, L. Peel, C. Rafferty, F. Simutis, and A. Harrill. Methodological considerations for measuring biofluid-based microRNA biomarkers. CRITICAL REVIEWS IN TOXICOLOGY. Taylor & Francis Group, London, UK, 51(3): 264-282, (2021).
Diatom DNA sequence data
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The raw data consisted of demultiplexed fastq files pairs (R1.fastq and R2.fastq) per sample accessible on the NCBI Sequences Read Archive (SRA) under the BioProject accession numbers PRJNA1187555 for experiments E1 and E3 and PRJNA1187576 for E2 and E4. This dataset is associated with the following publication: Valentin, V., S. Rivera, E. Acs, S. Almeida, K. Andree, L. Apothéloz-Perret-Gentil, B. Bailet, A. Baričević, K. Beentjes, J. Bettig, A. Bouchez, C. Camilla, C. Chardon, M. Duleba, T. Elersek, C. Genthon, M. Jablonska, L. Jacas, M. Kahlert, M. Kelly, J. Macher, F. Mauri, M. Moletta-Denat, A. Mortágua, J. Pawlowski, J. Pérez-Burillo, M. Pfannkuchen, E. Pilgrim, P. Panayiota, F. Rimet, K. Stanic, K. Tapolczai, S. Theroux, R. Trobajo, B. Van der Hoorn, M. Vasquez, M. Vidal, D. Wanless, J. Warren, J. Zimmermann, and B. Paix. Proficiency testing and cross-laboratory method comparison to support standardisation of diatom DNA metabarcoding for freshwater biomonitoring. Metabarcoding and Metagenomics. Pensoft Publishers, Sofia, BULGARIA, e133264, (2025).
Use of qPCR and RT-qPCR for Monitoring Variations of Microcystin Producers and Early Warning Their Toxin Production in an Ohio Inland Lake
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qPCR and RT-qPCR. This dataset is associated with the following publication: Lu, J., I. Struewing, L. Wymer, D. Tettenhorst, J. Shoemaker, and J. Allen. Use of qPCR and RT-qPCR for monitoring variations of microcystin producers and as an early warning system to predict toxin production in an Ohio inland lake. WATER RESEARCH. Elsevier Science Ltd, New York, NY, USA, 170: 115262, (2020).
Ceratocystis lukuohia spore dilution for probit analysis
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Environmental DNA (eDNA) detection tools are becoming increasingly popular for documenting occurrence and distribution of native and invasive species. These tools can allow early detection of new diseases and invasive species and provide critical information for land management. We designed two new samplers for monitoring airborne particulates, including fungal and fern spores and plant pollen, that rely on natural wind currents (Passive Environmental Sampler) or a battery operated fan (Active Environmental Sampler). This dataset contains results of an experiment that was designed to determine probability of detecting known numbers of Ceratocystis lukuohia spores on individual slides in these samplers.
Taking control of microplastics data: A comparison of control/blank data correction methods
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Establishing a robust strategy to account for unintended processing contamination in microplastics research is of interest to the microplastic community who are currently focussed on developing harmonised methods, and to environmental managers who are calling for accurate risk assessment wrt microplastics. Six commonly used 'core' data correction methods were assessed for their suitablity to microplastics research: a) No correction; b) Subtraction; c) Subtraction of mean; d) Spectral Similarity; e) Limits of detection/ limits of quantification (LOD/LOQ) and f) Statistical analysis. An additional 45 variant methods based on these 6 core methods (n=51 in total) were used to correct a dummy microplastics dataset using control data. The dummy microplastics dataset (n=10 identical samples) was created in the laboratory to mimic the laboratory contamination which may arise throughout sample processing and handling. These were free from ‘sample’ matrix but contained processing solutions and MilliQ water as the surrogate sample matrix. Microplastics processing was conducted following AIMS Microplastics SOPs, and polymer type identified by FTIR and confirmed against the Nicodom Polymer Library. Data was analysed in Excel and R. Bayesian analysis was also assessed for suitablity. This work informs work practices for the IMOS long-term microplastic monitoring project, and for all projects conducted by the AIMS microplastics group. This work will also inform the wider microplastics community by starting the conversation towards harmonisation of microplastics data analysis and reporting.
Datasets for "Early microRNA responses in rodent liver mediated by furan exposure establish dose thresholds for later adverse outcomes" manuscript
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These data are associated with the figures and tables presented in the manuscript "Early microRNA responses in rodent liver mediated by furan exposure establish dose thresholds for later adverse outcomes". Each spreadsheet contains the data for each Figure and metadata describes each column and/or row of data. For access to the smallRNA-seq files, please follow link provided and supply the reviewer key code "mjsnewymzlollmz".