Comparison of microRNA Profiling Platforms (NanoString)
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ABSTRACT: Global miRNA expression profiling of human malignancies is gaining popularity in both basic and clinically driven research. But to date, the majority of such analyses have used microarrays and quantitative real-time PCR. With the introduction of digital count technologies, such as next-generation sequencing (NGS) and the NanoString nCounter System, we have at our disposal, many more options. To make effective use of these different platforms, the strengths and pitfalls of several miRNA profiling technologies were assessed, including a microarray platform, NGS technologies and the NanoString nCounter System. These results were compared to gold-standard quantitative real-time PCR. Comparison of non-small cell lung cancer cell lines grown in vitro (n = 5) and in vivo (n = 5) as xenograft models.
Project description:Global miRNA expression profiling of human malignancies is gaining popularity in both basic and clinically driven research. But to date, the majority of such analyses have used microarrays and quantitative real-time PCR. With the introduction of digital count technologies, such as next-generation sequencing (NGS) and the NanoString nCounter System, we have at our disposal, many more options. To make effective use of these different platforms, the strengths and pitfalls of several miRNA profiling technologies were assessed, including a microarray platform, NGS technologies and the NanoString nCounter System. These results were compared to gold-standard quantitative real-time PCR. Comparison of non-small cell lung cancer cell lines grown in vitro (n = 5) and in vivo (n = 5) as xenograft models.
Project description:Background: Molecular adaptations in the striatum mediated by dopamine (DA) denervation or Levodopa (L-dopa) treatment have been implicated with the motor deficits found in Parkinsonâ??s disease (PD). Alterations in glutamatergic neurotransmission and anti-oxidant mechanisms are reported to play important roles in mediating these changes. However, the mechanisms mediating the molecular adaptations in the striatum are not well understood. In recent years, microRNAs (miRNAs) have been recognized as potent post-transcriptional regulators of gene expression with fundamental roles in numerous biological processes. miRNAs are known to influence the development and maintenance of striatal neurons. Therefore, we sought to determine the genome-wide expression levels of miRNAs in PD striatal tissues. Methods: Using a digital gene expression platform to quantify miRNA levels, we compared the expression of 800 miRNAs in human postmortem putamen tissues from PD patients and controls. Results: We detected the expression of approximately 250 miRNAs in postmortem human putamen samples collected from patients with PD and healthy controls. There was an abundance of a subset of 17 miRNAs (10 up- and 7 down-regulated) differing substantially between PD and the control tissues. Conclusions: We identified deregulated miRNAs most likely associated with altered striatal functions found in PD. This approach may provide insight into pathogenesis and additional therapeutic targets for the development novel treatment strategies for the disease. Human postmortem putamen tissues from 12 patients with PD symptoms and 12 neurologically normal controls. Samples were obtained from the Human Brain and Spinal Fluid Resource Center, Los Angeles, CA through NIH NeuroBioBank, and stored at -80°C until RNA isolation. Total RNA was extracted using the miRVana RNA isolation kit, following the manufacturerâ??s instructions (Ambion). Using 225ng total RNA, miRNA levels were assayed by direct digital detection using Nanostring miRNA assay kits (Nanostring Technologies).
Project description:In this study, breast cancer MCF-7 cells were cultured under 0.5% oxygen for 24 h followed by 24 h of reoxygenation. Cells was harvested at 0, 1, 12, and 24 h during reoxygenation, and examined the miRNA profile by Nanostring nCounter. Forty-three miRNAs had dramatic changes as compared with 0 h upon reoxygenation, with 63% (n=27) of the miRNAs up-regulated upon reoxygenation. Among these miRNAs, miR-769-3p, which was down-regulated in MCF-7 upon reoxygenation, was chosen for further investigation. The hypothesis was that the down-regulation of NDRG1 upon reoxygenation was regulated by miRNAs. To examine wether miRNAs regulate NDRG1 under 0.5% O2 concentrations, the miRNA expression profiles of MCF-7 cells under reoxygenation were examined. Cells were harvested at 0 (hypoxia control), 1, 12, and 24 h upon reoxygenation. Each profile was done in triplicate. The genomic profile of miRNAs was measured using NanoString nCounter® miRNA Expression Assays.
Project description:Background: Molecular adaptations in the striatum mediated by dopamine (DA) denervation or Levodopa (L-dopa) treatment have been implicated with the motor deficits found in Parkinsonâs disease (PD). Alterations in glutamatergic neurotransmission and anti-oxidant mechanisms are reported to play important roles in mediating these changes. However, the mechanisms mediating the molecular adaptations in the striatum are not well understood. In recent years, microRNAs (miRNAs) have been recognized as potent post-transcriptional regulators of gene expression with fundamental roles in numerous biological processes. miRNAs are known to influence the development and maintenance of striatal neurons. To determine the contribution of miRNAs in molecular adaptations found in PD, we screened the expression of 800 miRNAs in postmortem striatum samples obtained from PD and neurologically normal controls. We detected the expression of approximately 250 miRNAs in postmortem human putamen samples collected from patients with PD and healthy controls. There was an abundance of a subset of 17 miRNAs (10 up- and 7 down-regulated) differing substantially between PD and the control, suggesting that miRNAs are altered in the striatum during the course of PD. Computational analysis show that many of these miRNAs targets pro-inflammatory factors in the striatum. Therefore, we sought to detrmine the expression of experimentally validated pro-inflammatory transcripts in PD striatum. Methods: Using a digital gene expression platform to quantify 134 gene transcripts, we compared human postmortem putamen tissues from patients with PD and neurologically normal controls. Results: We identified deregulated expression of 10 gene transcripts (4 up- and 6 down-regulated mRNAs) in postmortem human putamen samples collected fromPD patients compared to controls. The expression of these genes negative correlates with the expression of many of the differentially epxressed miRNAs. Moreover, many of these genes are predicted targets of the differentially expressed miRNAs. Conclusions: We identified deregulated mRNAs most likely associated with anti-oxidant mechanims in PD striatum. This approach may provide insights into pathogenesis of the disease. Human postmortem putamen tissues from 12 patients with PD symptoms and 12 neurologically normal controls were used in the study. Samples were obtained from the Human Brain and Spinal Fluid Resource Center, Los Angeles, CA through NIH NeuroBioBank, and stored at -80°C until RNA isolation. Total RNA was extracted using the mirVana RNA isolation kit, following the manufacturerâs instructions (Ambion). Using 225ng total RNA, mRNA levels were assayed by direct digital detection using Nanostring nCounter assay kits (Nanostring Technologies).
Project description:Emerging evidence suggests that circulating exosomes mediate some of the beneficial effects of exercise via the transfer of microRNAs between tissues. However, the impact of short-term (0.5 year in this study) and long-term (25+ years in this study) regular bouts of exercise on circulating exosomal microRNAs (exomiRs) remains unknown. Our aim was to elucidate the prevention pattern of exomiRs and their targeted pathways. Therefore, in the present study we analyzed serum exomiR expression in healthy young, sedentary participants (n=14) at baseline and following a half year-long moderate-intensity regular exercise training. We also analyzed serum exomiR expression in older, healthy trained participants (seniors, n=11) who engaged in endurance activities for at least 25 years. Following the isolation and enrichment of serum exosomes their exomiR levels were determined using the amplification-free Nanostring platform.
Project description:Variation in individuals' responses to environmental factors is believed to influence susceptibility to complex diseases in humans. The genetic basis of such variation is poorly understood. We measured gene expression from resting and stimulated dendritic cells (DCs) derived from the peripheral blood of healthy individuals. We stimulated the primary DCs with E. coli lipopolysaccharide (LPS), influenza virus or the cytokine IFNβ, and associated genetic variation between individuals with the observed variation in gene expression and gene induction. We collected peripheral blood from each human donor. We isolated peripheral blood mononuclear cells by Ficoll, and magnetically sorted them for CD14+CD16- monocytes. We then differentiated the monocytes into monocyte-derived dendritic cells (MoDCs) by culturing the cells for 7 days with GM-CSF and IL-4. We stimulated the cells with E. coli lipopolysaccharide (LPS) for 2.5 hr or 5 hr, influenza (PR8 dNS1) for 10 hr, or recombinant IFN-beta for 6.5 hr. Finally, we lysed the cells and ran Nanostring on the lysates.
Project description:In this study, we performed miRNA profiles analysis of 84, 17, 20, 83, 718, 1123, 528 and 816 glioma spheres compared to normal progenitor sample (16wf) using microarray to evaluate their potential role in regulation of the biological properties of proneural and mesenchymal glioma spheres miRNA profiling analysis of the 9 samples including 8 patient-derived samples including glioma sphere samples (84, 17, 20, 718, 816, 528, 83 and 1123), as well as one normal progenitor sample (16wf).
Project description:The most frequent mature aggressive B-cell lymphomas are diffuse large B-cell lymphoma (DLBCL) and Burkitt lymphoma (BL). Patients suffering from molecularly defined BL (mBL) but treated with a regimen developed for DLBCL show an unfavorable outcome compared to mBL treated with chemotherapy regimens for BL. Distinguishing BL from DLBCL by conventional histopathology is challenging in lymphomas that have features common to both diseases (aggressive B-cell lymphoma unclassifiable with features of DLBCL and BL [intermediates]). Moreover, DLBCL are a heterogeneous group of lymphomas comprising distinct molecular subtypes: the activated B-cell (ABC)-like, the germinal center B-cell-like (GCB) and the unclassifyable subtype as defined by gene expression profiling (GEP). Attempts to replace GEP with techniques applicable to formalin-fixed paraffin-embedded (FFPE) tissue led to algorithms for immunohistochemical stainings (IHS). Disappointingly, the algorithms yielded conflicting results with respect to their prognostic potential, raising concerns about their validity. Furthermore, IHS algorithms did not provide a fully resolved classification: They did not identify mBL; nor did they separate ABC from unclassified DLBCL. 29 diffuse large B-Cell lymphoma samples were hybridized to HGU133A Affymetrix GeneChips. In addition, this study contains 22 already published samples whereas 11 of them contribute to GSE22470, 6 contribute to GSE10172, 3 to GSE44164 and 2 to GSE4475. No re-normalisation of published samples was performed. We used digital multiplexed gene expression (DMGE) with FFPE derived RNA to classify agressive B-cell lymphomas. Our assay comprised only 30 genes (10 for the detection of mBL and 20 for the detection of ABC and GCB). We chose these genes by reanalysis of the microarray data reported in a previous study. 39 samples from mature aggressive B-cell lymphomas were analyzed using DMGE (nCounter, NanoString Technologies Inc., Seattle, WA, USA) of FFPE- and fresh-frozen derived RNA. All cases were previously characterized by the Molecular Mechanisms of Malignant Lymphoma (MMML) consortium using the Affymetrix GeneChip technology (gold standard of classification). Please note that there are total 40 FFPE-derived and 50 fresh-frozen derived samples, with 39 samples derived from both materials (allowing direct comparison).