Plasma miRNA Expression Profiling Using Human TaqMan Low Density Array Cards
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
SUBMITTER: Koichi Murata
PROVIDER: E-GEOD-46012 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:miRNA expression profiling was performed on MM.1S MM cells cultured 8 hours in control media or 50nM RGB-286638, with or without BMSCs. The emerging role of miRNAs in the pathogenesis of multiple myeloma (MM) led us to hypothesize that the miRNA network might be among the inducible transcriptional alterations consequent to MM-bone marrow stromal cell (BMSC) interactions. Our data suggests that BMSC induced MM transcription led to aberrant miRNA expression. We therefore hypothesized that agents interfering with RNAPII transcription might inhibit aberrant miRNA expression in MM. To test this hypothesis we used RGB-286638, a novel protein kinase inhibitor, which works primarily via RNAPII inhibition followed by transcriptional arrest in MM cells. miRNA profiling of RGB-286638-exposed MM cells resulted in RNAPII arrest associated with reduced miRNA levels. RGB-286638 abrogated BMSCs-induced miRNAs, which correlated with growth arrest in MM cells. Analysis of RGB-286638-induced differentially-expressed miRNAs in MM cells, in the presence or absence of BMSCs, revealed RNAPII regulation of expression of BMSC-inducible miRNAs with established oncogenic functions in MM Our findings demonstrate the role of RNAPII in regulating miRNA network, suggesting a new rationale for using agents interfering with RNAPII transcription in the treatment of MM. TaqMan Low-Density Array (TLDA) using human miRNA version 2.0A and version 3.0B cards (Applied Biosystems) were applied to examine the global change of miRNA expression levels in MM.1S cells when co-cultured with BMSCs, with or without RGB-286638 treatment. A total of 756 mature miRNA updated in the Sanger miRBase v.15.0 were quantified according to the manufacturer's instructions as previously described. miRNAs with Ct values higher than 37 were excluded from the analysis. Normalization was carried out with the mean of RNU44 and RNU48. Relative quantification of miRNA expression was calculated with the 2M-bM-^HM-^RM-NM-^TM-NM-^TCt Ct method using the ddCt program (Shannon McCormack Advanced Molecular Diagnostics Laboratory Research Services). The data was presented as log10 of the relative quantity of each miRNA.
2012-05-31 | E-GEOD-38364 | biostudies-arrayexpress
Project description:MicroRNAs (miRNAs) are non-coding RNAs that play a fundamental role in regulation of gene expression affecting differentiation and development. In particular, miRNAs have been described to regulate genes important for pancreatic development and islet function. The aim of this work was to determine the miRNA expression signature in human pancreatic alpha and beta cells. miRNA stability to fixation allowed the study of microRNA in pure populations of human alpha and beta cells sorted by FACS after intracellular staining with glucagon and insulin, respectively. The determination of the specific group of miRNAs expressed in the human pancreatic alpha and beta cells may further the understanding of gene expression regulation of the islet differentiation process. The alpha and beta cells come from 6 different preparations of human pancreatic islets from donors. In this study we define expression profiles of a total of 665 miRNAs for pancreatic alpha and beta cells. For this purpose, cells were fixed with paraformaldehyde, 7AAD was applied to exclude dead cells. Then, cells were sorted after intracellular staining with C peptide to detect beta cells and glucagon to detect alpha cells. After sorting, we confirmed enriched beta cells have a purity of on average over 98%. Enriched alpha cells have a purity of on average over 98%. To determine the miRNA expression profiles, we used human miRNA TLDAs version 2. For each sample card A and card B were run after cDNA synthesis and 12 cycles of preamplification according to the manufacturer protocol. Each TLDA card A contains 1 probe for the endogenous control RNU48 while each TLDA card B contains 4 replicates of the RNU48 probe. Analysis of these controls allows calculating the intra- and inter-assay variation. Quantitative values (RQ) were calculated measuring the ddCt between the Ct values of each miRNA and the Ct value of the small nucleolar RNU48 RNA comparing the target sample and the control sample.
2013-01-30 | E-GEOD-38360 | biostudies-arrayexpress
Project description:MicroRNA expression profiling during muscle stem cell activation. Quiescent muscle stem cells from uninjured muscles and activated muscle stem cells from injured muscles at indicated time points were isolated by FACS. MicroRNA expression profiling during muscle stem cell activation using real-time PCR based miRNA arrays. Muscle stem cells were harvested at indicated time points (0hr, 36hr, 60hr and 72hr) after injury. 3 technical replicates were performed. Supplementary files: Raw data (Ct) and complete processed data (dCt, ddCt, fold-change) by platform.
2012-01-01 | E-GEOD-26780 | biostudies-arrayexpress
Project description:Signal transducer and activator of transcription 3 (STAT3) is a critical transcription factor in cancer. However, while the protein-coding target genes of STAT3 have been extensively studied, the microRNA target genes of STAT3 are less understood. MicroRNAs are short, non-coding RNAs that regulate messenger RNAs through translational inhibition and transcript degradation. They have been found to be involved in all aspects of cancer biology. Given the roles of both STAT3 and miRNAs in cancer, the function of STAT3 as a transcription factor, and the dearth of known STAT3 miRNA targets, our goal was to identify novel STAT3 miRNA targets relevant to cancer. To do so, we engineered MCF-10A cells with doxycycline-inducible expression of STAT3C. STAT3C is a constitutively-active mutant version of STAT3. Although STAT3 can be activated by various growth factors and kinases, other pathways can be activated as well, which would confound analysis of the results. Thus, the advantage of STAT3C is that it allowed specific and focused activation of STAT3 alone, and this screen represents the first genome-wide survey of miRNA expression changes associated specifically with STAT3 activity. MCF-10A, a non-transformed breast epithelial cell line, was chosen because STAT3C has been reported to be sufficient to cause their neoplastic transformation. Therefore, we reasoned that analysis of STAT3C’s effects in MCF-10A cells would be especially informative for STAT3-regulated miRNAs relevant to cancer. As a result of our study, we identified previously-known as well as novel miRNA targets of STAT3. Doxycycline-inducible MCF-10A cells were seeded, and then untreated (grown in standard growth media alone) or treated with 2?g/ml doxycycline for 48hr. Each condition was performed in biological triplicate (labeled A, B, and C), for a total of 6 samples. Total RNA was harvested and submitted to the Dana-Farber Cancer Institute Molecular Diagnostics Laboratory. MicroRNA expression profiling was performed using TaqMan Low-Density Arrays (TLDA), human miRNA version 2.0A and version 3.0B cards (Applied Biosystems).
2014-02-05 | E-GEOD-44089 | biostudies-arrayexpress
Project description:MicroRNAs (miRNAs) are non-coding RNAs that play a fundamental role in regulation of gene expression affecting differentiation and development. In particular, miRNAs have been described to regulate genes important for pancreatic development and islet function. The aim of this study was to determine the miRNA expression signature during human pancreas development. We identified 212 miRNAs that were expressed throughout different gestational ages. From those, 4 miRNAs increased (group I), 35 miRNAs decreased (group II), during pancreatic development. The expression of the remaining 173 miRNAs was relatively constant throughout all assessed gestational ages. The determination of the specific group of miRNAs expressed in the human developing pancreas may further the understanding of gene expression regulation during this important process. In this study we define expression profiles of a total of 352 miRNAs for different stages of the pancreas development. Each TLDA card contains 2 endogenous controls which are present in 8 replicates each. Analysis of these controls allows calculating the intra- and inter-assay variation. Quantitative values (RQ) were calculated measuring the dCt between the Ct values of each miRNA and the Ct value of the small nucleolar RNU48 RNA.
2010-09-06 | E-GEOD-22026 | biostudies-arrayexpress
Project description:Human milk (breastmilk) is much more than nutrition for the infant, containing an array of regulatory agents with immunoprotective and developmental functions. Amongst those, microRNAs (miRNAs) have recently been identified, with their properties, roles, origin and distribution in breastmilk as well as in the mammary gland being still undetermined. In this study, we examined the miRNA profile of different fractions of human milk (cells and lipids) using the OpenArray system (Applied Biosystems, 770 miRNA species measured per sample) and compared it with maternal peripheral blood mononuclear cells (PBMCs) and plasma. Although PBMCs were the richest group in miRNA species, plasma showed very low expression pattern. Thus, the human milk fractions (cells, lipid) and skim milk (not being investigated in this study) were found to conserve higher levels of miRNAs than blood in general. Specifically, human milk cell miRNA quantity was found relatively close to PBMCs, and higher than milk lipids. Correlation and clustering analyses indicated that miRNA expression and types of milk cells were highly similar to those in lipids. Milk miRNAs showed a slight correlation to PBMCs, so PBMCs potentially are not contributing to milk miRNAs. Plasma was different to all other three groups in miRNA content and expression pattern. Further, two infant formulae (a plant-based and a cow milk-based) were compared to human milk and found to contain significantly fewer miRNA species than human milk cells and lipids (p>0.001). Taken together with previous studies on miRNAs, our findings demonstrate that human milk is one of the richest sources of miRNAs among human body fluids. As a non-invasive and plentiful source of miRNAs, human milk could be used as a disease biomarker for the mammary gland, with potential in assessing lactation performance. Finally, gene target and pathways analyses identified several target mRNAs regulated by miRNAs found to be abundant in breastmilk. Given the recently identified stability and function of food-derived miRNAs in regulating mammalian genes, we propose that breastmilk is a rich source of miRNA ingested by the infant during the first months of life, and which potentially contribute to early infant development. 10 exclusively breastfeeding dyads were recruited. 10 whole milk and 10 whole blood samples were collected and fractionated to obtain 10 milk cells, 10 milk lipid, 10 mononeucleoted blood cells (PBMCs), and 10 plasma. In addition to the above 40 samples, 2 infant formula were profiled. 4 different extraction kits were used, miRNeasy mini Kit for human milk cell and PBMC samples. miRCURY RNA Isolation-Biofluids Kit for human milk lipid samples and both infant formulae. mirVana PARIS Kit for plasma samples. NanoDrop 2000 and Bioanalyzer 2100 were used to determine concentration and purity of the extracted miRNA from all samples (n=42). miRNA OpenArray panel system (Life Technologies, CA, USA) was used to profile 754 human mature miRNAs in samples. RNU48, RNU44 and U6 rRNA were used as housekeeping controls for normalisation. ath-miR159a was used as a negative control for human samples. GeneGO and Ingenuity Pathway Analysis were used to determine biological pathways. Please note that normalization of miRNAs was done in R but without generating deltaCT values, thus [1] only the list of normalized miRNA with Ct vlaue between 8 and 29 and that detected in at least 4 samples out of 10 analysed in each group is provided ('normalized_miRNAs_list.txt') [2] the sample data tables contain raw data.
2015-03-30 | E-GEOD-66358 | biostudies-arrayexpress
Project description:Dysregulated miRNA in human colorectal cancer (CRC) were identified through comparison between 4 CRC tumors and their adjacent normal tissues by miRNA array. Histologically-confirmed CRC were included in this study. CRC tissues and paired adjacent normal tissues were obtained from the resected surgical specimens. The adjacent normal tissue is composed of normal colonic mucosa located at approximately 10 cm away from the cancer tissue. miRNA profiling of 754 human miRNAs was performed using TaqMan Human MiRNA Array Set v3.0. Quantitative real-time polymerase chain reaction (Q-PCR) was performed using Applied Biosystems 7900HT Real-Time PCR System (Applied Biosystems). Results were analyzed by the SDS RQ Manager 1.2 software (Applied Biosystems). 4 CRC tissues and 4 adjacent normal tissues were subjucted to qPCR based miRNA expression profiling. Equal amount of total RNA were used for analysis.
2013-03-20 | E-GEOD-45349 | biostudies-arrayexpress
Project description:MicroRNAs (miRNAs) regulate activity of protein-coding genes including those involved in hematopoietic cancers. The goal of the current study was to explore which miRNAs are unique for seven different subtypes of pediatric acute lymphoblastic leukemia (ALL). Therefore, the expression levels of 397 miRNAs (including novel miRNAs) were measured by quantitative RT-PCR in 81 pediatric leukemia cases and 17 normal hematopoietic control cases. Except for BCR-ABL-positive and B-other ALL, all major subtypes i.e. T-ALL, MLL-rearranged, TEL-AML1-positive, E2A-PBX1-positive and hyperdiploid ALL have unique miRNA-signatures that differ from each other and from those in healthy hematopoietic cells. Strikingly, the miRNA signature between TEL-AML1-positive and hyperdiploid cases partly overlapped, which suggests a common underlying biology. Moreover, aberrant downregulation of let-7b (~70-fold) in MLL-rearranged ALL was linked to upregulation of oncoprotein c-Myc (P<0.0001). Besides genetic aberrations, in vitro drug resistance predicts clinical outcome. Resistance to vincristine and daunorubicin was characterized by ~20-fold upregulation of miR-125b, miR-99a and miR-100 (P≤0.002). No discriminative miRNAs were found for prednisolone and only one miRNA was linked to L-asparaginase resistance. Finally we show that the expression levels of 14 miRNAs were --independently of subtype-- associated with clinical outcome in pediatric ALL. We conclude that genetic subtypes and drug resistant leukemic cells display characteristic miRNA signatures in pediatric ALL. Functional studies of discriminative and prognostic important miRNAs may provide new insights into the biology of disease. Experiment type: stem-loop real-time (RT) quantitative PCR (RT-qPCR) Bone marrow and peripheral blood samples were collected from children at newly diagnosis of acute lymphoblastic leukemia (ALL). CD34+ -cells were sorted from G-CSF-stimulated blood cell samples of children with a brain tumor or Wilm’s tumor. Thymocytes were isolated from thymic lobes that were resected from children during surgery for congenital heart disease. RNA from the cell samples was extracted using TRIzol reagents (firma). Only RNA with an RNA Integrity Number (RIN) of ≥ 7.5 as measured by the 2100 Bioanalyzer (Agilent, Amstelveen, the Netherlands) was used as input for the RT-qPCR reactions. All miRNAs were validated with the stem-loop real-time (RT) quantitative PCR (RT-qPCR) technique by using either TaqMan MicroRNA Array MicroRNA arrays (v 1.0, Early Access) or Custom TaqMan MicroRNA Array arrays (Applied Biosystems, Foster City, USA). Values represent ∆Ct in each individual patient for whom the specific subtype, identification number and cellular drug-resistance is shown on top of each column. Drug-resistance was based on median LC50 values (concentration of a drug lethal to 50% of the leukemic cells) that have reported prognostic impact in children with newly diagnosed ALL. Median LC50 values were used to assign patients as sensitive (≤ median LC50) or resistant (> median LC50) to the drug in question. The ∆Ct value was calculated according to the following equation: Ct value of the specific miRNA minus the Ct value of the internal reference. In case of the TaqMan MicroRNA Array MicroRNA arrays the mean Ct values for snoR-13 and snoR-14 were used as reference whereas in case of the Custom TaqMan MicroRNA Array arrays snoR-1 was used. MiRNAs validated by the TaqMan MicroRNA Array MicroRNA arrays are listed together with the part numbers of the corresponding (stem-loop) primers as available by Applied Biosystems, Foster City, USA. Primers for the remaining miRNAs were custom designed by Applied Biosystems. Abbreviations: MLL: MLL-rearranged precursor B-ALL, B-other: precursor B-ALL negative for MLL-rearrangements, TEL-AML1, BCR-ABL, E2A-PBX and hyperdiploidy (> 50 chromosomes). CD34+: normal CD34+-sorted blood progenitor cells, nBM: normal bone marrow.
2011-02-11 | E-GEOD-23024 | biostudies-arrayexpress
Project description:MicroRNAs (miRNAs) are important in the regulation of many biological processes such as growth and development. To evaluate the role of miRNAs in skeletal muscle regeneration, global miRNA expression was measured during muscle cell growth and differentiation. Primary cultures of murine myogenic progenitor cells (MPC) were studied for miRNA expression using quantitative PCR-array. During MPC differentiation or proliferation, 139 or 16 miRNAs, respectively, exhibited significant >2-fold changes. Cluster analysis revealed 5 distinct miRNA expression patterns at different stages of differentiation. Fourteen miRNAs exhibiting >10-fold change during differentiation included miR-1, 10b, 96, 98, 133a, 139-5p, 330, 335-3p, 339-5p, 344, 486, 499, 504, and 598. Ten of these miRNAs were located in introns of protein coding genes, such as miR-499 located in the myosin heavy chain isoform Myh7b. In silico analysis of possible miRNA-mRNA interactions indicated that many of these miRNAs targeted mRNA critically involved in muscle differentiation. Interestingly, several miRNAs targeted different sites in a given mRNA, suggesting coordinated expression of multiple miRNAs to ensure the regulation of essential genes. These results identify differentially expressed miRNAs that could represent new regulatory elements in MPC proliferation and differentiation. Myogenic progenitor cell (MPC) growth and differentiation are key elements duing muscle regeneration. Using defined culture conditions to promote proliferation or differentiation, we profiled miRNA expression in primary cultures of murine MPC.
2009-06-13 | E-GEOD-16473 | biostudies-arrayexpress
Project description:Eosinophlic esophagitis (EoE) is increasely recognized as an antigen-drived disorder. The goal of this study is to reveal the miRNA expression changes in EoE before and after a successful glucocorticoid steroid treatment. Total RNA was extracted from the esophageal epithelial layers of 5 paired paraffin-embedded biopsies before and after treatment with glucocorticosteroids using RecoverAll Total Nucleic Acid Extraction Kit for FFPE tissues (Ambion, Austin, TX). Five nanograms of total RNA was reverse-transcribed using the Taqman MicroRNA Reverse Transcription Kit and the Megaplex RT primer Human Pool A (Applied Biosystems). The reverse-transcribed cDNA was then pre-amplified in 12 cycles of PCR using Taqman PreAmp Master Mix and the Megaplex PreAmp primers, Human Pool A (Applied Biosystems). The cDNA’s were then diluted and loaded on to a Taqman Human miRNA Array card A (Platform GPL9731 ; Applied Biosystems), which contains probes for 377 distinct miRNAs. The Array cards were run on an ABI HT7900 qPCR instrument. Ct values were obtained for all miRNAs represented on the cards and fold changes in expression were calculated using the delta delta Ct (ddCt) method.
2012-03-23 | E-GEOD-36726 | biostudies-arrayexpress