Comparison of miRNA quantitation by Nanostring in serum and plasma samples.
ABSTRACT: Circulating microRNAs that are associated with specific diseases have garnered much attention for use in diagnostic assays. However, detection of disease-associated miRNA can be affected by several factors such as release of contaminating cellular miRNA during sample collection, variations due to amplification of transcript for detection, or controls used for normalization for accurate quantitation. We analyzed circulating miRNA in serum and plasma samples obtained concurrently from 28 patients, using a Nanostring quantitative assay platform. Total RNA concentration ranged from 32-125 ?g/ml from serum and 30-220 ?g/ml from plasma. Of 798 miRNAs, 371 miRNAs were not detected in either serum or plasma samples. 427 were detected in either serum or plasma but not both, whereas 151 miRNA were detected in both serum and plasma samples. The diversity of miRNA detected was greater in plasma than in serum samples. In serum samples, the number of detected miRNA ranged from 3 to 82 with a median of 17, whereas in plasma samples, the number of miRNA detected ranged from 25 to 221 with a median of 91. Several miRNA such as miR451a, miR 16-5p, miR-223-3p, and mir25-3p were highly abundant and differentially expressed between serum and plasma. The detection of endogenous and exogenous control miRNAs varied in serum and plasma, with higher levels observed in plasma. Gene expression stability identified candidate invariant microRNA that were highly stable across all samples, and could be used for normalization. In conclusion, there are significant differences in both the number of miRNA detected and the amount of miRNA detected between serum and plasma. Normalization using miRNA with constant expression is essential to minimize the impact of technical variations. Given the challenges involved, ideal candidates for blood based biomarkers would be those that are indifferent to type of body fluid, are detectable and can be reliably quantitated.
Project description:Circulating microRNAs (ci-miRNAs) in blood have emerged as promising diagnostic, prognostic and predictive biomarkers in cancer. Many clinical studies currently incorporate studies that assess ci-miRNAs. Validation of the clinical significance of candidate biomarker miRNAs has proven to be difficult, potentially resulting from vast discrepancies in the detection methodology as well as biological variability. In the current study, the influence of several methodological factors on ci-miRNA detection was evaluated as well as short-term biological variability in patients with head and neck cancer. RNA was isolated from 124 serum and plasma samples originating from patients with head and neck cancer and healthy volunteers. The miRNA levels were measured using RT-qPCR and the influence of pre-analytical factors, different normalization strategies and temporal reproducibility was assessed. RNA carriers improved ci-miRNA detection in serum and plasma specimens. A prolonged pre-processing time correlated with an increased hemolytic index in serum samples only. Hemolysis differentially affected the detection of individual miRNAs. Optimal normalization was achieved using the averaged detection values of spike-in cel-miR-39-3p and endogenous miR-16-5p. Comparing biological replicates from patients with head and neck cancer, the intra-individual miRNA levels were relatively stable (average interval 7 days). Differences in the ci-miRNA detection methodology and limitations of currently used technologies can greatly affect the results and may explain inconsistent outcomes between studies. Prior to the implementation of ci-miRNAs as useful clinical biomarkers, further advances in the standardization of the detection methodology and reduction of technical variability are needed.
Project description:Novel noninvasive biomarkers with high sensitivity and specificity for the diagnosis of breast cancer (BC) are urgently needed in clinics. The aim of this study was to explore whether miRNAs from the miR-106a-363 cluster can be detected in the circulation of BC patients and whether these miRNAs can serve as potential diagnostic biomarkers.The expression of 12 miRNAs from the miR-106a-363 cluster was evaluated using qRT-PCR in 400 plasma samples (from 200 BC patients and 200 healthy controls (HCs)) and 406 serum samples (from 204 BC patients and 202 HCs) via a three-phase study. The identified miRNAs were further examined in tissues (32 paired breast tissues), plasma exosomes (from 32 BC patients and 32 HCs), and serum exosomes (from 32 BC patients and 32 HCs).Upregulated levels of four plasma miRNAs (miR-106a-3p, miR-106a-5p, miR-20b-5p, and miR-92a-2-5p) and four serum miRNAs (miR-106a-5p, miR-19b-3p, miR-20b-5p, and miR-92a-3p) were identified and validated in BC. A plasma 4-miRNA panel and a serum 4-miRNA panel were constructed to discriminate BC patients from HCs. The areas under the receiver-operating characteristic curves of the plasma panel were 0.880, 0.902, and 0.858, and those of the serum panel were 0.910, 0.974, and 0.949 for the training, testing, and external validation phases, respectively. Two overlapping miRNAs (miR-106a-5p and miR-20b-5p) were consistently upregulated in BC tissues. Except for the expression of the plasma-derived exosomal miR-20b-5p, the expression patterns of exosomal miRNAs were concordant between plasma and serum, indicating the potential use of exosomal miRNAs as biomarkers.We identified four plasma miRNAs and four serum miRNAs from the miR-106a-363 cluster as promising novel biomarkers for the diagnosis of BC.
Project description:BACKGROUND:Serum biomarkers for diagnosis and risk stratification of childhood solid tumors would improve the accuracy/timeliness of diagnosis and reduce the need for invasive biopsies. We hypothesized that differential expression and/or release of microRNAs (miRNAs) by such tumors may be detected as altered serum miRNA profiles. METHODS:We undertook global quantitative reverse transcription PCR (qRT-PCR) miRNA profiling (n = 741) on RNA from 53 serum samples, representing 33 diagnostic cases of common childhood cancers plus 20 controls. Technical confirmation was performed in a subset of 21 cases, plus four independent samples. RESULTS:We incorporated robust quality control steps for RNA extraction, qRT-PCR efficiency and hemolysis quantification. We evaluated multiple methods to normalize global profiling data and identified the 'global mean' approach as optimal. We generated a panel of six miRNAs that were most stable in pediatric serum samples and therefore most suitable for normalization of targeted miRNA qRT-PCR data. Tumor-specific serum miRNA profiles were identified for each tumor type and selected miRNAs underwent confirmatory testing. We identified a panel of miRNAs (miR-124-3p/miR-9-3p/miR-218-5p/miR-490-5p/miR-1538) of potential importance in the clinical management of neuroblastoma, as they were consistently highly overexpressed in MYCN-amplified high-risk cases (MYCN-NB). We also derived candidate miRNA panels for noninvasive differential diagnosis of a liver mass (hepatoblastoma vs. combined MYCN-NB/NB), an abdominal mass (Wilms tumor vs. combined MYCN-NB/NB), and sarcoma subtypes. CONCLUSIONS:This study describes a pipeline for robust diagnostic serum miRNA profiling in childhood solid tumors, and has identified candidate miRNA profiles for prospective testing. IMPACT:We propose a new noninvasive method with the potential to diagnose childhood solid tumors.
Project description:Our objective was to identify suitable reference genes in serum miRNA for normalization and screen potential new biomarkers for osteoporosis diagnosis by a systematic study. Two types of osteoporosis models were used like as mechanical unloading and estrogen deficiency. Through a large-scale screening using microarray, qPCR validation and statistical algorithms, we first identified miR-25-3p as a suitable reference gene for both type of osteoporosis, which also showed stability during the differentiation processes of osteoblast and osteoclast. Then 15 serum miRNAs with differential expression in OVX rats were identified by microarray and qPCR validation. We further detected these 15 miRNAs in postmenopausal women and bedrest rhesus monkeys and evaluated their diagnostic value by ROC analysis. Among these miRNAs, miR-30b-5p was significantly down-regulated in postmenopausal women with osteopenia or osteoporosis; miR-103-3p, miR-142-3p, miR-328-3p were only significantly decreased in osteoporosis. They all showed positive correlations with BMD. Except miR328-3p, the other three miRNAs were also declined in the rhesus monkeys after long-duration bedrest. Their AUC values (all >0.75) proved the diagnostic potential. Our results provided a reliable normalization reference gene and verified a group of circulating miRNAs as non-invasive biomarkers in the detection of postmenopausal- and mechanical unloading- osteoporosis.
Project description:BACKGROUND:The consensus on how to choose a reference gene for serum or plasma miRNA expression qPCR studies has not been reached and none of the potential candidates have yet been convincingly validated. We proposed a new in silico approach of finding a suitable reference for human, circulating miRNAs and identified a new set of endogenous reference miRNA based on miRNA profiling experiments from Gene Expression Omnibus. We used 3 known normalization algorithms (NormFinder, BestKeeper, GeNorm) to calculate a new normalization score. We searched for a universal set of endogenous miRNAs and validated our findings on 2 new datasets using our approach. RESULTS:We discovered and validated a set of 13 miRNAs (miR-222, miR-92a, miR-27a, miR-17, miR-24, miR-320a, miR-25, miR-126, miR-19b, miR-199a-3p, miR-30b, miR-30c, miR-374a) that can be used to create a reliable reference combination of 3 miRNAs. We showed that on average the mean of 3 miRNAs (p =?0.0002) and 2 miRNAs (p =?0.0031) were a better reference than single miRNA. The arithmetic means of 3 miRNAs: miR-24, miR-222 and miR-27a was shown to be the most stable combination of 3 miRNAs in validation sets. CONCLUSIONS:No single miRNA was suitable as a universal reference in serum miRNA qPCR profiling, but it was possible to designate a set of miRNAs, which consistently contributed to most stable combinations.
Project description:Adult-onset Still's disease (AOSD) is a systemic inflammatory disease characterized by cytokine storm. However, a diagnostic test for AOSD in clinical use is yet to be validated. The aim of our study was to identify non-invasive biomarkers with high specificity and sensitivity to diagnosis of AOSD. MicroRNA (miRNA) profiles in PBMC from new-onset AOSD patients without any treatment and healthy controls (HCs) were analyzed by miRNA deep sequencing. Plasma samples from 100 AOSD patients and 60 HCs were used to validated the expression levels of miRNA by qRT-PCR. The correlations between expression levels of miRNAs and clinical manifestations were analyzed using advanced statistical models. We found that plasma samples from AOSD patients showed a distinct miRNA expression profile. Five miRNAs (miR-142-5p, miR-101-3p, miR-29a-3p, miR-29c-3p, and miR-141-3p) were significantly upregulated in plasma of AOSD patients compared with HCs both in training and validation sets. We discovered a panel including 3 miRNAs (miR-142-5p, miR-101-3p, and miR-29a-3p) that can predict the probability of AOSD with an area under the receiver operating characteristic (ROC) curve of 0.8250 in training and validation sets. Moreover, the expression levels of 5 miRNAs were significantly higher in active AOSD patients compared with those in inactive patients. In addition, elevated level of miR-101-3p was found in AOSD patients with fever, sore throat and arthralgia symptoms; the miR-101-3p was also positively correlated with the levels of IL-6 and TNF-? in serum. Furthermore, five miRNAs (miR-142-5p, miR-101-3p, miR-29c-3p, miR-29a-3p, and miR-141-3p) expressed in plasma were significantly higher in AOSD patients than in sepsis patients (P < 0.05). The AUC value of 4-miRNA panel (miR-142-5p, miR-101-3p, miR-29c-3p, and miR-141-3p) for AOSD diagnosis from sepsis was 0.8448, revealing the potentially diagnostic value to distinguish AOSD patients from sepsis patients. Our results have identified a specific plasma miRNA signature that may serve as a potential non-invasive biomarker for diagnosis of AOSD and monitoring disease activity.
Project description:<h4>Background</h4>Recently a large number of short non-coding-RNAs (microRNAs, (miRNA)) have been identified. These miRNAs act as post-transcriptional regulators where they generally have an inhibitory function. miRNAs are present in all human cells, and they are also detected in serum or plasma. The miRNAs have a broad range of actions, and their biogenesis must therefore be under tight control. One putative regulator of miRNA biogenesis or miRNA level could be vitamin D, an ancient hormone with effects on cell growth and differentiation, apoptosis and the immune system. In our study miRNA were reversed transcribed in total RNA isolated from plasma and analyzed by quantitative real-time PCR (qPCR) using the miRCURY LNA Universal RT microRNA PCR system (Exiqon). In 10 pilot subjects 136 miRNAs were detected in one or more plasma samples drawn at baseline and after 12 months of vitamin D supplementation. The twelve miRNAs that showed the greatest change in expression in these pilots were further analyzed by RT-qPCR of RNA from baseline and 12 months plasma samples in 40 subjects given high dose vitamin D(3) (20.000-40.000 IU per week) and 37 subjects given placebo.<h4>Results</h4>At baseline there was a significant and positive correlation between serum 25-hydroxyvitamin D and miR-532-3p expression (r = 0.24, P = 0.04). The change in expression of miR-221 from baseline to 12 months (ddCp value) was also significantly different between the vitamin D and placebo group (P =0.04), mainly due to a change in the placebo group.<h4>Conclusions</h4>We have not been able to demonstrate a consistent effect of vitamin D supplementation on the expression profile of miRNA in plasma. However, further studies are needed as this approach might potentially throw light on unknown aspects of vitamin D physiology.
Project description:Pancreatic cancer (PC) has posed a great health threat to a growing number of people all over the world. Detection of serum miRNAs, being sensitive, noninvasive, and easy to obtain, has a great potential of being a novel screening method for PC patients. In this study, we investigated miRNA expression levels in serum by qRT-PCR. The study was divided into four phases: the screening, training, testing, and external validation stage. We firstly chose candidate miRNAs using Exiqon panels in the screening phase. Then, a total of 129 PC serum samples and 107 normal controls (NCs) were further analyzed in the following training and testing phases to identify differently expressed miRNAs. A cohort of 30 PC serum samples vs 30 NCs was used to confirm the diagnostic value of the identified miRNAs in the external validation phase. Moreover, miRNA expressions in additional 44 PC tumor tissue samples and the matched adjacent normal tissue samples as well as 32 pairs of serum-derived exosomes samples were also further explored. As a result, we identified six significantly upregulated miRNAs in the serum of PC: let-7b-5p, miR-192-5p, miR-19a-3p, miR-19b-3p, miR-223-3p, and miR-25-3p. A six-miRNA panel in serum was then established. The area under the receiver operating characteristic curves (AUC) for the panel was 0.910 for the combined training and testing phases, which showed higher diagnostic value than the individual miRNA. Prognostic value prediction using Cox's proportional hazards model and Kaplan-Meier curves showed that increased serum miR-19a-3p was closely related to worse overall survival (OS). In addition, significant upregulation of miR-192-5p, miR-19a-3p, and miR-19b-3p was observed in both PC tissue and serum-derived exosomes samples. In conclusion, we identified a six-miRNA (let-7b-5p, miR-192-5p, miR-19a-3p, miR-19b-3p, miR-223-3p, and miR-25-3p) panel in the serum for PC early and noninvasive diagnosis.
Project description:The outstanding characteristics of circulatory microRNAs (miRNAs) attract much attention in research on disease biomarkers and disease pathogenesis. This study aimed to identify the expression profiles of plasma miRNAs in patients with rheumatoid arthritis (RA). Thirty-three miRNAs were screened using an miRNA array, of which 9 miRNAs were validated as differentially expressed in the plasma of RA patients compared with healthy controls (HCs). miRNA-4634 (miR-4634), miR-181d and miR-4764-5p expression levels were increased, whereas miR-342-3p, miR-3926, miR-3925-3p, miR-122-3p, miR-9-5p and miR-219-2-3p expression levels were decreased in RA patients. The areas under the curve (AUCs) were generated to estimate the sensitivity and specificity of each miRNA or the panel of all 9 miRNAs as biomarkers for RA. AUCs for 9 individual miRNAs ranged from 0.6254 to 0.818; however, the AUC for the panel of 9 miRNAs reached 0.964. Levels of miR-122-3p, miR-3925-3p, miR-342-3p and miR-4764-5p expression showed significant differences between RA and other control groups. miR-4764-5p, miR-4634, miR-9-5p and miR-219-2-3p exhibited significant correlations with either plasma cytokine and chemokine levels or clinical features. In conclusion, this study identified 9-plasma miRNAs signature in Chinese patients with RA which may serve as noninvasive biomarkers for the diagnosis of RA.
Project description:<h4>Background</h4>MicroRNAs (miRNAs) are small RNAs that regulate gene expression by suppressing protein translation and may influence RNA expression. MicroRNAs are detected in extracellular locations such as plasma; however, the extent of miRNA expression in plasma its relation to cardiovascular disease is not clear and many clinical studies have utilized array-based platforms with poor reproducibility.<h4>Methods and results</h4>Initially, to define distribution of miRNA in human blood; whole blood, platelets, mononuclear cells, plasma, and serum from 5 normal individuals were screened for 852 miRNAs using high-throughput micro-fluidic quantitative RT-PCR (qRT-PCR). In total; 609, 448, 658, 147, and 178 miRNAs were found to be expressed in moderate to high levels in whole blood, platelets, mononuclear cells, plasma, and serum, respectively, with some miRNAs uniquely expressed. To determine the cardiovascular relevance of blood miRNA expression, plasma miRNA (n=852) levels were measured in 83 patients presenting for cardiac catheterization. Eight plasma miRNAs were found to have over 2-fold increased expression in patients with significant coronary disease (?70% stenosis) as compared to those with minimal coronary disease (less than 70% stenosis) or normal coronary arteries. Expression of miR-494, miR-490-3p, and miR-769-3p were found to have significantly different levels of expression. Using a multivariable regression model including cardiovascular risk factors and medications, hsa-miR-769-3p was found to be significantly correlated with the presence of significant coronary atherosclerosis.<h4>Conclusions</h4>This study utilized a superior high-throughput qRT-PCR based method and found that miRNAs are found to be widely expressed in human blood with differences expressed between cellular and extracellular fractions. Importantly, specific miRNAs from circulating plasma are associated with the presence of significant coronary disease.