ABSTRACT: In this study, we aim to identify common miRNA signatures in the pathogenesis of different NMD groups (Duchenne Muscular Dystrophy, Megaconial Congenital Muscular Dystrophy (CMD), Ullrich CMD and alpha-dystroglycanopathy) (abbreviated as D, M, U, and A, respectively) each caused by mutations in different genes encoding proteins with distinct roles. For this purpose, we isolated miRNAs from the skeletal muscle tissues of three patients from four disease groups and three control individuals (15 individuals in total) and we performed miRNA microarray method (Affymetrix GeneChip miRNA 4.0 Array). In order to find out differentially expressed miRNAs in patients, we analyzed raw data by two different databases. Differentially expressed miRNAs that were found to be statistically significant by using both the programs (with parameters, fold change ≥2.0 and FDR=0 for MeV-SAM analysis; and p<0.05 for TAC-ANOVA analysis) were identified as the potential miRNA candidates.
Project description:Global gene expression analysis was performed comparing human skeletal muscle samples from patients with various forms of muscular dystrophy and mitochondrial myopathies in order to identify specific gene expression changes associated with collagen VI deficiency (leading to Ullrich´s Congenital Muscular Dystrophy) and depletion of mitochondrial DNA relative to other mitochondrial myopathies We analysed the gene expression profile of skeletal muscle from children suffering from mitochondrial myopathies and various forms of muscular dystrophy relative to skeletal muscle from healthy children using commercially available arrays that represents the complete human genome (Agilent Human SurePrintGE, 8x60K )
Project description:26 limb-girdle muscular dystrophy patients from Latvia and 34 patients from Lithuania with clinical symptoms of limb-girdle muscular dystrophies, along with 204 healthy unrelated controls were genotyped for 96 most frequent known limb-girdle muscular dystrophies causing mutations for the region, using VeraCode GoldenGate system. More information can be found in article Robust genotyping tool for autosomal recessive type of limb-girdle muscular dystrophies in BMC Musculoskeletal Disorders by I. Inashkina et al.
Project description:This is a large series human Duchenne muscular dystrophy patient muscle biopsies, in specific age groups, using all available Affymetrix arrays (including a custom MuscleChip produced by the Hoffman lab). Both mixed groups of patients (5 patient biopsies per group) and individual biopsies were done. Hypothesis: That the progression of DMD can be understood in terms of muscle molecular remodeling. Keywords: other
Project description:The extraocular muscles (EOMs) are a unique group of muscles that are anatomically and physiologically distinct from other skeletal muscles. Previously, we and others have shown that EOMs have a unique transcriptome and proteome. Here, we investigated the expression pattern of microRNAs (miRNAs) in EOM, as they may play a role in generating the unique EOM allotype. We screened LC Sciences miRNA microarrays covering the sequences of miRBase 10.0 to define the microRNAome of normal mouse EOM and tibialis anterior (TA) limb muscle. 74 miRNAs were found to be differentially regulated (p-value < 0.05) and 31 miRNAs (14 up-regulated and 17 down-regulated) were found to be differentially regulated at a signal strength > 500 including the muscle-specific miR-206, miR-1, miR-133a, miR-133b and miR-499. qPCR analysis was used to validate the differential expression. Bioinformatic tools were used to identify potential miRNA-mRNA-protein interactions and integrate data with previous transcriptome and proteomic profiling data. Luciferase assays using co-transfection of precursor miRNAs (pre-miRNAs) along with reporter constructs containing the 3’-untranslated region (3’UTR) of their predicted target genes were used to validate targeting by identified miRNAs. The definition of the EOM microRNAome complements existing transcriptome and proteome data about the molecular make-up of EOM and provides further insight into regulation of muscle genes. These data will also help to further explain the unique EOM muscle allotype and its differential sensitivity to diseases such as Duchenne's muscular dystrophy (DMD) and may assist in development of therapeutic strategies. Total RNA from four EOM and four TA tissue samples dissected from four adult male C57/Bl10 mice were used (TA served as control) to screen four LC Sciences microRNA Microarray chips. The chips contained microRNA sequences based on miRBase content 10.0 totalling 568 different miRNAs. Samples were labelled with Cy3 and Cy5 using dye-swap. Relative differences of miRNA expression was expressed as fold-changes EOM/TA, which were calculated after normalization across all four arrays.
Project description:MicroRNAs (miRNAs) are small non-coding RNAs with strong biological functions. However, the roles of miRNAs in alcohol addiction are still unclear. In this study, we analyzed miRNA profile of in the nucleus accumbens (NAc) of rats treated with alcohol. The results demonstrated, among the 300 detected miRNAs in NAc, multiple miRNAs were aberrantly expressed after treatment with alcohol. To perform the experiment, 18 male rats (weighing 150-180 g) were divided into two treatment groups: vehicle (500 ul saline, ip bid) or alcohol (1 g/kg, ip bid). Seven days later, the animals were sacrificed and their NAc were isolated for miRNA microarray analysis. The miRNA expressions were determined using miRNA microarrays for rat (LC Sciences). Data were normalized using cyclic method and statistically analyzed using Ttest.
Project description:UB cells FACS sorted from E15.5 mouse kidneys were analyzed for miRNAs expression. E15.5 UB cells FACS sorted from Rosa26YFP; Hoxb7Cre mice, total RNAs including miRNAs were purified and used for miRNA microarray
Project description:MicroRNAs (miRNAs) are small noncoding RNAs that negatively regulate gene expression post-transcriptionally. They play a critical role in developmental and physiological processes and have been implicated in the pathogenesis of several diseases including cancer. To identify miRNA signatures associated with different stages of neoplastic development, we examined the expression profile of 776 primate miRNAs in the following cells: primary African green monkey kidney (pAGMK) cells; spontaneously immortalized, non-tumorigenic, low-passage VERO cells (10-87 LP); tumorigenic, high-passage VERO cells (10-87 HP); and a cell line (10-87 T) derived from a 10-87 HP cell tumor xenograft in athymic nude mice. When compared with pAGMK cells, the majority of miRNAs were expressed at lower levels in 10-87 LP, 10-87 HP, and 10-87 T cells. We identified 10 up-regulated miRNAs whose level of expression correlated with VERO cell evolution from a non-tumorigenic phenotype to a tumorigenic phenotype. Several miRNAs that were components of the tumorigenic phenotype-specific signatures in our AGMK model are also found in a variety of human tumors. This may prove to be of general relevance to the biology of neoplastic development as it occurs both in vivo as well as in vitro. In addition, one or more of these miRNAs could be potential biomarkers for the expression of the tumorigenic phenotype of VERO cells. The spontaneousely transformed VERO cells, non-tumorigenic, were pasasged at low density in culture up to 250. The high passage (p250) was found to be tumorigenic. The cell line from xenograft of high passage was also established. We then evaluated patterns of miRNA expression in pAGMK cells and in derivatives of the 10-87 VERO cell line (10-87 LP cells, 10-87 HP cells, and 10-87 T cells) in an attempt to identify the miRNAs whose altered expression might correlate with, and perhaps be involved in, the evolution of the neoplastic phenotypes that occurred during passage of these AGMK cells in tissue culture. performed high-throughput miRNA profiling to audit the expression level of miRNAs in pAGMK cells and in VERO cells at non-tumorigenic and tumorigenic stages of neoplastic development. The analysis involved pAGMK cells, non-tumorigenic 10-87 low-passage VERO cells (10-87 LP) tumorigenic, high-passage VERO cells (10-87 HP) and a cell line (10-87 T) derived from a 10-87 HP cell tumor xenograft in athymic nude mice.
Project description:Objectives: The collagen VI related muscular dystrophies (COL6-RD), Ullrich congenital muscular dystrophy (UCMD) and Bethlem myopathy (BM) are among the most common congenital muscular dystrophies, but the pathogenesis, including the role of mutant collagen VI in the matrix is poorly understood. To better define the pathways disrupted by mutations in collagen VI, we have used a transcriptional profiling approach with RNA-Seq to identify differentially expressed genes in COL6-RD patients from controls. Methods: We have used RNA-Seq to identify differentially expressed genes in cultured dermal fibroblasts from 13 COL6-RD patients (8 dominant negative and 5 null) and 6 controls. Sequence reads were analyzed using the TopHat/Cufflinks pipeline. Results: Differentially expressed transcripts between COL6-RD patient and control fibroblasts include upregulation of ECM components and downregulation of factors controlling matrix remodeling and repair. DN and null samples are differentiated by downregulation of genes involved with DNA replication and repair in null samples Overall design: Expression profiles of dermal fibroblasts from 13 COL6-RD patients with dominant negative (8) or null (5) mutations compared to 6 control fibroblasts.
Project description:High reproducibility with TaqMan microRNA array (qPCR-array) was demonstrated by comparing replicate results from the same RNA sample. Pre-amplification of the miRNA cDNA improved sensitivity of the qPCR-array and increased the number of detectable miRNAs. Furthermore, the relative expression levels of miRNAs were maintained after pre-amplification. When the performance of qPCR-array and microarrays were compared using different aliquots of the same RNA, a low correlation between the two methods (r = -0.443) indicated considerable variability between the two assay platforms. Higher variation between replicates was observed in miRNAs with low expression in both assays. Finally, a higher false positive rate of differential miRNA expression was observed using the microarray compared to the qPCR-array. Replicate preparations (n =4) using different aliquots of a single C2C12 RNA sample (500 ng) were used to determine the reproducibility of the reverse transcription process as well as the results of the same reverse transcription products performed on different days. TaqMan microRNA Array A was used for this purpose. To assess the reliability of pre-amplification, miRNA expression profiles obtained with and without pre-amplification using MiRNA TaqMan Array B were performed. Independent miRNA expression profiling studies using uParaflo microfluidic biochips were performed by an independent company to determine the relationship between results obtained with qPCR-array and microarrays. Aliquots of the same C2C12 RNA were used in both the qPCR-array (500 ng) and microarrays (8 µg).
Project description:miRNA profiling of human tonsilar B lymphocytes (naïve B cells, centroblasts and memory B cells) and tonsilar T cells, analyzing the 470 human miRNAs present in miRBase V9.1. B cell subpopulations and T cells were purified using specific antibodies, magnetic beads and the AutoMACS system (Miltenyi Biotec) from reactive tonsils obtained from routine tonsilectomies. Informed consents and the Institutional Review Board approval were obtained. Total RNA was extracted from cell pellets using the mirVana™ miRNA Isolation Kit (Ambion), and sent to LC Sciences facility for microarray hybridization using microfluidics technology. FirstChoice® Human Skeletal Muscle Total RNA (Ambion) was used as common reference for all hybridizations. Background substracted and normalized data from each channel was used to calculate log ratios sample/reference. Keywords: miRNA profiling Dual channel experiments using FirstChoice® Human Skeletal Muscle Total RNA (Ambion) as common reference for all hybridizations. Reference was labeled with Cy5 and the lymphocyte extracted RNA with Cy3. Biological replicates: 4 for each B cell subpopulation (centroblasts, naïve and memory B cells) and 3 for T cells, all of them purified from reactive tonsils with specific antibodies and magnetic beads. Microarray hybridization was performed at LC Sciences, using slides containing in situ synthesized oligonucleotides to detect the 470 miRNAs present in miRBase V9.1. Probes hsa-miR-337 and hsa-miR-542-5p were excluded due to systematic dye bias, according to the manufacturer intructions. Background substracted and normalized detectable data was used to calculate log ratios sample/reference. Background is determined using a regression-based background mapping method. The regression is performed on 5% to 25% of the lowest intensity data points excluding blank spots. Raw data matrix is then subtracted by the background matrix. Normalization was carried out using a LOWESS (Locally-weighted Regression) method on the background-subtracted data. A transcript to be listed as detectable must meets at least two conditions: signal intensity higher than 3×(background standard deviation) and spot CV < 0.5. CV is calculated by (standard deviation)/(signal intensity). When repeating probes are present on an array, a transcript is listed as detectable only if the signals from at least 50% of the repeating probes are above detection level.