Plasma microrna quantification protocol
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ABSTRACT:
SUBMITTER: Maiocchi S
PROVIDER: S-EPMC10914336 | biostudies-literature | 2023 Jan
REPOSITORIES: biostudies-literature
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Project description:The first step in biomarkers discovery is to identify the best protocols for their purification and analysis. We have identified an optimal RNA extraction method of microRNAs from human plasma samples. We also report that the addition of low doses of carrier RNA before starting RNA extraction improves microRNA extraction and quantification.
2013-10-18 | GSE51375 | GEO
Project description:The first step in biomarkers discovery is to identify the best protocols for their purification and analysis. We have identified an optimal RNA extraction method of microRNAs from human plasma samples. We also report that the addition of low doses of carrier RNA before starting RNA extraction improves microRNA extraction and quantification. Human plasma and matched biopsies were obtained from healthy donors and patients attended at the Hospital Universitari i Politècnic La Fe (Valencia, Spain). RNA was extracted by different preanalytal conditions and reagents, testing the suitable of carrier addition at differnt doses. The best protocol was followed up by hybridation on Affymetrix microarrays.
2013-10-18 | E-GEOD-51375 | biostudies-arrayexpress
Project description:ObjectiveCirculating microRNAs are promising diagnostics and prognostics biomarkers in a wide variety of diseases. However, there is a critical reproducibility challenge, which in part may be due to preanalytical factors. MicroRNA purification has been identified as the major contributor to the total intra assay variation, thus we found great interest in recent papers describing methods for direct quantification of circulating microRNAs without the purification step. With one exception, all the studies we identified where a direct quantification of circulating microRNAs had been performed were using SYBR Green chemistry. In our laboratory we use platelet-poor plasma and TaqMan assays for microRNA analysis, and thus we investigated whether we could adapt the procedures for the direct reverse transcription described by these studies to be used with our TaqMan assays.ResultsWe did not achieve valid results by direct quantification of selected microRNAs (miR-92a, miR-16 and miR-126) in platelet-poor plasma using TaqMan assays.
| S-EPMC6509816 | biostudies-literature
Project description:Extracellular vesicles (EV) containing microRNAs (miRNAs) have tremendous potential as biomarkers for the early detection of disease. Here, we present a simple and rapid PCR-free integrated microfluidics platform capable of absolute quantification (<10% uncertainty) of both free-floating miRNAs and EV-miRNAs in plasma with 1 pM detection sensitivity. The assay time is only 30 minutes as opposed to 13 h and requires only ~20 μL of sample as oppose to 1 mL for conventional RT-qPCR techniques. The platform integrates a surface acoustic wave (SAW) EV lysing microfluidic chip with a concentration and sensing microfluidic chip incorporating an electrokinetic membrane sensor that is based on non-equilibrium ionic currents. Unlike conventional RT-qPCR methods, this technology does not require EV extraction, RNA purification, reverse transcription, or amplification. This platform can be easily extended for other RNA and DNA targets of interest, thus providing a viable screening tool for early disease diagnosis, prognosis, and monitoring of therapeutic response.
| S-EPMC6527557 | biostudies-literature
Project description:The first step in biomarkers discovery is to identify the best protocols for their purification and analysis. This issue is critical when considering peripheral blood samples (plasma and serum) that are clinically interesting but meet several methodological problems, mainly complexity and low biomarker concentration. Analysis of small molecules, such as circulating microRNAs, should overcome these disadvantages. The present study describes an optimal RNA extraction method of microRNAs from human plasma samples. Different reagents and commercially available kits have been analyzed, identifying also the best pre-analytical conditions for plasma isolation. Between all of them, the column-based approaches were shown to be the most effective. In this context, miRNeasy Serum/Plasma Kit (from Qiagen) rendered more concentrated RNA, that was better suited for microarrays studies and did not require extra purification steps for sample concentration and purification than phenol based extraction methods. We also present evidences that the addition of low doses of an RNA carrier before starting the extraction process improves microRNA purification while an already published carrier dose can result in significant bias over microRNA profiles. Quality controls for best protocol selection were developed by spectrophotometry measurement of contaminants and microfluidics electrophoresis (Agilent 2100 Bioanalyzer) for RNA integrity. Selected donor and patient plasma samples and matched biopsies were tested by Affymetrix microarray technology to compare differentially expressed microRNAs. In summary, this study defines an optimized protocol for microRNA purification from human blood samples, increasing the performance of assays and shedding light over the best way to discover and use these biomarkers in clinical practice.
| S-EPMC3871541 | biostudies-literature
Project description:Numerous studies have reported a potential role for circulating microRNAs as biomarkers in a wide variety of diseases. However, there is a critical reproducibility challenge some of which might be due to differences in preanalytical and/or analytical factors. Thus, in the current study we systematically investigated the impact of selected preanalytical and analytical variables on the measured microRNA levels in plasma. Similar levels of microRNA were found in platelet-poor plasma obtained by dual compared to prolonged single centrifugation. In contrast, poor correlation was observed between measurements in standard plasma compared to platelet-poor plasma. The correlation between quantitative real-time PCR and droplet digital PCR was found to be good, contrary to TaqMan Low Density Array and single TaqMan assays where no correlation could be demonstrated. Dependent on the specific microRNA measured and the normalization strategy used, the intra- and inter-assay variation of quantitative real-time PCR were found to be 4.2-6.8% and 10.5-31.4%, respectively. Using droplet digital PCR the intra-assay variation was 4.4-20.1%, and the inter-assay variation 5.7-26.7%. Plasma preparation and microRNA purification were found to account for 39-73% of the total intra-assay variation, dependent on the microRNA measured and the normalization strategy used. In conclusion, our study highlighted the importance of reporting comprehensive methodological information when publishing, allowing others to perform validation studies where preanalytical and analytical variables as causes for divergent results can be minimized. Furthermore, if microRNAs are to become routinely used diagnostic or prognostic biomarkers, the differences in plasma microRNA levels between health and diseased subjects must exceed the high preanalytical and analytical variability.
| S-EPMC6053236 | biostudies-literature
Project description:Background. Various microRNAs (miRNAs) are used as markers of acute coronary syndrome, in which heparinization is considered mandatory therapy. Nevertheless, a standard method of handling plasma samples has not been proposed, and the effects of heparin treatment on miRNA detection are rarely discussed. Materials and Method. This study used quantitative polymerase chain reaction (qPCR) analysis to investigate how storage temperature, standby time, hemolysis, and heparin treatment affect miRNA measurement in plasma samples from 25 patients undergoing cardiac catheterization. Results. For most miRNAs, the qPCR results remained consistent during the first 2 hours. The miRNA signals did not significantly differ between samples stored at 4°C before processing and samples stored at room temperature (RT) before processing. miR-451a/miR-23a ratio < 60 indicated < 0.12% hemolysis with 100% sensitivity and 100% specificity. Pretreatment with 0.25?U heparinase I recovered qPCR signals that were reduced by in vivo heparinization. Conclusions. For miRNA measurement, blood samples stored at RT should be processed into plasma within 2 hours after withdrawal and should be pretreated with 0.25?U heparinase I to overcome heparin-attenuated miRNA signals. The miR-451a/miR-23a ratio is a reliable indicator of significant hemolysis.
| S-EPMC5048034 | biostudies-literature
Project description:BackgroundDifferent technologies, such as quantitative real-time PCR or microarrays, have been developed to measure microRNA (miRNA) expression levels. Quantification of miRNA transcripts implicates data normalization using endogenous and exogenous reference genes for data correction. However, there is no consensus about an optimal normalization strategy. The choice of a reference gene remains problematic and can have a serious impact on the actual available transcript levels and, consequently, on the biological interpretation of data.ContentIn this review article we discuss the reliability of the use of small RNAs, commonly reported in the literature as miRNA expression normalizers, and compare different strategies used for data normalization.SummaryA workflow strategy is proposed for normalization of miRNA expression data in an attempt to provide a basis for the establishment of a global standard procedure that will allow comparison across studies.
| S-EPMC4890630 | biostudies-literature
Project description:BackgroundSecondary brain injury accounts for a major part of the morbidity and mortality in patients with spontaneous aneurysmal subarachnoid hemorrhage (SAH), but the pathogenesis and pathophysiology remain controversial. MicroRNAs (miRNAs) are important posttranscriptional regulators of complementary mRNA targets and have been implicated in the pathophysiology of other types of acute brain injury. Cerebral microdialysis is a promising tool to investigate these mechanisms. We hypothesized that miRNAs would be present in human cerebral microdialysate.MethodsRNA was extracted and miRNA profiles were established using high throughput real-time quantification PCR on the following material: 1) Microdialysate sampled in vitro from A) a solution of total RNA extracted from human brain, B) cerebrospinal fluid (CSF) from a neurologically healthy patient, and C) a patient with SAH; and 2) cerebral microdialysate and CSF sampled in vivo from two patients with SAH. MiRNAs were categorized according to their relative recovery (RR) and a pathway analysis was performed for miRNAs exhibiting a high RR in vivo.ResultsSeventy-one of the 160 miRNAs detected in CSF were also found in in vivo microdialysate from SAH patients. Furthermore specific miRNAs consistently exhibited either a high or low RR in both in vitro and in vivo microdialysate. Analysis of repeatability showed lower analytical variation in microdialysate than in CSF.ConclusionsMiRNAs are detectable in cerebral microdialysate; a large group of miRNAs consistently showed a high RR in cerebral microdialysate. Measurement of cerebral interstitial miRNA concentrations may aid in the investigation of secondary brain injury in neurocritical conditions.
| S-EPMC4438475 | biostudies-literature
Project description:Background:Psoriasis is an immune-mediated inflammatory chronic skin disease characterized by chronic inflammation in the dermis, parakeratosis, and excessive epidermal growth. MicroRNAs (miRNAs) are key regulators of immune responses. Although differential expression of miRNAs has been reported in certain inflammatory autoimmune diseases, their role in psoriasis has not been fully illuminated. Our aims were to confirm plasma miRNA expression signatures in psoriasis and to examine their potential influence on psoriasis pathogenesis. Methods:A miRNome PCR array was used to analyse the plasma of psoriasis patients and healthy donors. We performed miRNA pathway enrichment and target gene network analyses on psoriasis plasma samples. Results:We found several specific plasma miRNA signatures relevant to psoriasis. The miRNAs targeted pathways associated with psoriasis, such as the VEGF, MAPK, and WNT signaling pathways. Network analysis revealed pivotal deregulated plasma miRNAs and their relevant target genes and pathways regulating psoriasis pathogenesis. Conclusions:This study analysed the expression of plasma miRNAs and their target pathways, elucidating the pathogenesis of psoriasis; these results may be used to design novel therapeutic strategies and to identify diagnostic biomarkers for psoriasis.
| S-EPMC7201701 | biostudies-literature