High resolution microRNA signatures in human whole saliva
Ontology highlight
ABSTRACT: Recent studies have reported both mRNA and microRNA (miRNA) in saliva, but little information has been documented on the quality and yield of RNA collected. Therefore, the aim of the present study was to develop an improved RNA isolation method from saliva and to identify major miRNA species in human whole saliva. RNA samples were isolated from normal human saliva using a combined protocol based on the Oragene RNA collection kit and the mirVana miRNA isolation kit in tandem. RNA samples were analyzed for quality and subjected to miRNA array analysis. Twelve representing 6 males and 6 females, were selected for miRNA profiling using the TaqManM-BM-. Low Density Array Card (TLDA) Human miRNA Panel v2.0 (Applied Biosystems). The analysis of expression of the >700 miRNAs was performed by the DNA Core at the Interdisciplinary Center for Biotechnology Research Center at the University of Florida, according to the manufacturerM-bM-^@M-^Ys protocol except the pre-amplification step was omitted. The NormFinder algorithm was used to identify the optimal normalization of miRNA among the 25 most abundantly expressed miRNAs detected.
Project description:MicroRNA expression profiling in matched lesional skin samples from 25 patients with psoriasis using the miRNA analysis platform miRCURY LNATM MicroRNA array (v. 11.0) (Exiqon). Aim: To explore the effect of three different preservation methods (Formalin-fixation paraffin-embedding (FFPE), frozen (FS) and OCT-embedding (OCT)) on miRNA expression levels in matched lesional skin samples from 25 patients with psoriasis. Three-condition experiment, FS vs. FFPE, OCT vs. FFPE and FS vs. OCT. Biological replicates: 25 matched samples from patients with psoriasis. One replicate per array.
Project description:miRNA expression profiles for round spermatids of wild type and GRTH knock-out mice were determined by Rodent TaqMan® Low Density miRNA Arrays A v2.0 (TLDA, Applied Biosystem). Purified round spermatids were prepared from the testis of wild type and GRTH null mice (C57BL/6 strain). Equal amount of total RNA from 20 mice (wild type or GRTH KO) was pooled prior to gene expression analysis.
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.
Project description:Simple steatosis (SS) and non-alcoholic steatohepatitis (NASH) are subtypes of non-alcoholic fatty liver disease. The difference in pathogenesis between SS and NASH is still not clear. MicroRNAs (miRNAs) are endogenous, non-coding short RNAs that regulate gene expression. The aim of this study was to examine the relationship of miRNA expression profiles with SS and NASH in animal models and humans. Animal models DD Shionogi (DS), Fatty Liver Shionogi (FLS Wild; W), and FLS ob/ob mice were subjected as the normal control, SS model and NASH model, respectively. Male DS mice (Riken BRC No. 03706) were provided by Riken bio-resource center through the national bio-resource project of Japan. Male FLS W and male FLS-ob/ob mice were obtained from Shionogi research laboratories (Shiga, Japan). Animals were housed in a room maintained at a controlled temperature of 24 ± 2 Ë?C under a 12-h light-dark cycle. Animals were provided ad libitum access to water and standard pellet feed. Five male mice of every strain (24 weeks old; mean body weight of DS, FLS W and FLS-ob/ob were 31.1 ± 1.1g, 38.4 ± 3.4g and 56.0 ± 5.0g, respectively) were sacrificed under pentobarbital anesthesia by whole blood collection from the right ventricle. The livers were cut into about 200 mg pieces and fixed in 10% formalin for histological analysis or fresh-frozen in liquid nitrogen and stored at -80 °C in a freezer until use. miRNA expression analysis Total RNAs in mouse liver were isolated using TrizolReagent (Life Technologies, California, USA) as described the manufacturerâ??s protocol. The quality of total RNA samples was checked by applying the RNA Integrity Number (RIN) which is calculated by a proprietary algorithm of the Agilent 2100 Bioanalyzer expert software (Agilent technologies, CA, USA). Only high quality RNA, with RNA integrity number (RIN) greater than 8 and A260/280 and A260/230 greater than 1.8, will be considered for microarray analysis. TaqMan® Array Rodent MicroRNA A Card v2.0 (Thermo Fisher Scientific, MA, USA) was used to assess 375 miRNA expression profiles in mouse liver tissue.
Project description:In order to identify aberrantly expressed microRNAs in oral squamous cell carcinomas (OSCCs), we have employed microRNA microarray profile using healthy tongue samples as control. All seventeen human OSCCs and three normal tongue tissues were collected from the Tissue Bank at the Moffitt Cancer Center and approved by the University of Florida institutional review board. Majority (15 out of 17) of the OSCCs were derived from tongue cancers. The tumor samples contained greater than 80% of cancerous cells, confirmed by microscopic examination by a head and neck pathologist. The normal tongues were taken from a non-cancerous region. Tissues were snap-frozen and stored at -80M-BM-0C until further use. Total RNAs were isolated from human tissues using mirVana miRNA Isolation kit (Ambion/Applied Biosystems, Austin, TX) according to the manufacturerM-bM-^@M-^Ys instruction. NanoDropM-BM-. ND-100 spectrophotometer (Thermo Scientific, Wilmington, DE) was used to quantify the isolated RNA. The Agilent 2100 Bioanalyzer from the Interdisciplinary Center for Biotechnology Research at the University of Florida was used to detect the size distribution of total RNA and to determine the quality as well. Expression of microRNAs from this signature was quantified in the same RNA samples by real-time PCR, confirming the expression pattern. Human tissues of oral squamous cell carcinoma and healthy normal tongues were used for microRNA microarray profile to identify aberrantly expressed microRNAs in oral squamous cell carcinomas.
Project description:Frozen blocks of human embryonic limb tissue were sectioned and laser capture microdissected to collect three human chodrocyte tissues - precursor, differentiated and hypertrophic chondrocytes.Total RNA including miRNAs was isolated and analyzed using Applied Biosystems OpenArrayM-BM-. Real-Time PCR system. RT-PCR miRNA expression profiling. miRNA expression from three chondrocyte sub types - precursor, differentiated and hypertrophic chondrocytes were analyzed using TaqManM-BM-. OpenArrayM-BM-. Human MicroRNA Panel.
Project description:Background: MicroRNAs (miRNAs) are small regulatory RNAs that are implicated in cancer pathogenesis and have recently shown promise as blood-based biomarkers for cancer detection. Epithelial ovarian cancer is a deadly disease for which improved outcomes could be achieved by successful early detection and enhanced understanding of molecular pathogenesis that leads to improved therapies. A critical step toward these goals is to establish a comprehensive view of miRNAs expressed in epithelial ovarian cancer tissues as well as in normal ovarian surface epithelial cells. Methodology: We used massively parallel pyrosequencing (i.e., M-bM-^@M-^\454 sequencingM-bM-^@M-^]) to discover and characterize novel and known miRNAs expressed in primary cultures of normal human ovarian surface epithelium (HOSE) and in tissue from three of the most common histotypes of ovarian cancer. Deep sequencing of small RNA cDNA libraries derived from normal HOSE and ovarian cancer samples yielded a total of 738,710 high-quality sequence reads, generating comprehensive digital profiles of miRNA expression. Expression profiles for 498 previously annotated miRNAs were delineated and we discovered six novel miRNAs and 39 candidate miRNAs. A set of 124 miRNAs was differentially expressed in normal versus cancer samples and 38 miRNAs were differentially expressed across histologic subtypes of ovarian cancer. Taqman qRT-PCR performed on a subset of miRNAs confirmed results of the sequencing-based study.
Project description:Cancer staging and treatment frequently assume a binary division of tumors into localized or metastatic cancers. We proposed a state of metastatic disease defined by the number of metastases termed oligometastases. Patients with oligometastatic disease may be cured with localized methods of cancer treatment. We analyzed miRNA expression from paraffin blocks of primary or metastatic tumor samples derived from oligometastatic (? 5 metastases) patients treated with high dose localized radiotherapy. We report patterns of miRNA expression in the metastatic and primary tumor samples that identify patients who failed to progress to widespread polymetastases. We created a model of oligometastases of human tumors in immune compromised mice. The miRNA patterns of gene expression derived from patients accurately identified oligometastatic patterns in the mouse model as compared to animals that developed widespread metastases. MiRNA signatures may identify patients most likely to benefit from aggressive curative treatment of limited metastatic disease. Injection of MDA-MB-435-GFP cancer cells into the mammary fat pad of female athymic mice to develop spontaneous macroscopic lung metastasis. Tail vein experimental lung colonization assay was performed to model the development of MDA-MB-435-GFP Oligo- or Poly-metastases in the lung in vivo. Cell lines: Total RNA were derived from MDA-MB-435-L1-GFP (Ol-like) or MDA-MB-435-L1Mic (Poly-like) cell lines.
Project description:Background: MicroRNA (miRNA) is an emerging subclass of small non-coding RNAs that regulates gene expression and has a pivotal role for many physiological processes including cancer development. Recent reports revealed the role of miRNAs as ideal biomarkers and therapeutic targets due to their tissue- or disease-specific nature. Head and neck cancer (HNC) is a major cause of cancer-related mortality and morbidity, and laryngeal cancer has the highest incidence in it. However, the molecular mechanisms involved in laryngeal cancer development remain to be known and highly sensitive biomarkers and novel promising therapy is necessary. Methodology/Principal Findings: To explore laryngeal cancer-specific miRNAs, RNA from 5 laryngeal surgical specimens including cancer and non-cancer tissues were hybridized to microarray carrying 723 human miRNAs. The resultant differentially expressed miRNAs were further tested by using quantitative real time PCR (qRT-PCR) on 43 laryngeal tissue samples including cancers, noncancerous counterparts, benign diseases and precancerous dysplasias. Significant expressional differences between matched pairs were reproduced in miR-133b, miR-455-5p, and miR-196a, among which miR-196a being the most promising cancer biomarker as validated by qRT-PCR analyses on additional 84 tissue samples. Deep sequencing analysis revealed both quantitative and qualitative deviation of miR-196a isomiR expression in laryngeal cancer. In situ hybridization confirmed laryngeal cancer-specific expression of miR-196a in both cancer and cancer stroma cells. Finally, inhibition of miR-196a counteracted cancer cell proliferation in both laryngeal cancer-derived cells and mouse xenograft model. Conclusions/Significance: Our study provided the possibilities that miR-196a might be very useful in diagnosing and treating laryngeal cancer. To explore laryngeal cancer-specific miRNAs, RNA from 5 laryngeal surgical specimens including cancer and non-cancer tissues were hybridized to microarray carrying 723 human miRNAs. Total RNA including low molecular weight RNA from tissue samples was isolated using the mirVanaTM miRNA Isolation Kit (Ambion) according to the manufacturer's instructions. The quality of the RNA samples was assessed using an Agilent 2100 Bioanalyzer, and only the samples meeting the criteria of 28S/18S > 1 and RNA Integrity Number (RIN) M-bM-^IM-% 7.5 were used for all analyses. For microarray analysis, we used the Human miRNA Microarray Kit V2 (Agilent Technologies, Santa Clara, CA), which contains 20-40 features targeting each of 723 human miRNAs (Agilent design ID 019118) as annotated in the Sanger miRBase, release 10.1. Labeling and hybridization of total RNA samples were performed according to the manufacturer's protocol. One hundred ng of total RNA was used as an input into the labeling reaction, and the entire reaction was hybridized to each array for 20 hours at 55M-BM-0C. The results were analyzed using Agilent GeneSpring GX7.3. Normal controls and cancer samples were compared using Welch's t-test (p<0.05) and differentially expressed miRNAs with at least a 2-fold change in expression were considered to be potential biomarkers.
Project description:MicroRNAs (miRNA) are short single-stranded RNA molecules that regulate gene expression post-transcriptionally by binding to complementary sequences in the 3' untranslated region (3' UTR) of target mRNAs. MiRNAs participate in the regulation of myogenesis, and identification of the complete set of miRNAs expressed in muscles is likely to significantly increase our understanding of muscle growth and development. To determine the identity and abundance of miRNA in porcine skeletal muscle, we applied a deep sequencing approach. This allowed us to identify the sequences and relative expression levels of 212 annotated miRNA genes, thereby providing a thorough account of the miRNA transcriptome in porcine muscle tissue. The expression levels displayed a very large range, as reflected by the number of sequence reads, which varied from single counts for rare miRNAs to several million reads for the most abundant miRNAs. Moreover, we identified numerous examples of mature miRNAs that were derived from opposite sides of the same predicted precursor stem-loop structures, and also observed length and sequence heterogeneity at the 5' and 3' ends. Furthermore, KEGG pathway analysis suggested that highly expressed miRNAs are involved in skeletal muscle development and regeneration, signal transduction, cell-cell and cell-extracellular matrix communication and neural development and function. Examination of small RNA profiles in 7 isolates of porcine muscle The raw sequences were trimmed to 30 nucleotides, and it was set as a requirement that any sequence must appear at least three times and be present in at least two of the seven libraries. All identical reads within a library were grouped and converted into unique sequences. Reads containing Ns or long tracks (M-BM-!M-CM-^]8) of As were removed and the sequences were trimmed for adaptor-sequences. To annotate the unique sequences, a Decypher Tera-BLASTN Search was performed against a database of mature miRNAs obtained from miRBase (release 12.0). Hits with a match of 16 or more nucleotides to a miRNA from the database were gathered, and the count of each miRNA was normalized to the total number of sequence reads per lane. The outcome of this procedure can be seen in the Aarhus_University_GBI_FC208D2AAXX_blast_mirbase table below.