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MicroRNA Profiling during Cardiomyocyte-Specific Differentiation of Murine Embryonic Stem Cells Based on Two Different miRNA Array Platforms  [PDF]
Lin Gan, Silke Schwengberg, Bernd Denecke
PLOS ONE , 2011, DOI: 10.1371/journal.pone.0025809
Abstract: MicroRNA (miRNA) plays a critical role in a wide variety of biological processes. Profiling miRNA expression during differentiation of embryonic stem cells will help to understand the regulation pathway of differentiation, which in turn may elucidate disease mechanisms. The identified miRNAs could then serve as a new group of possible therapeutic targets. In the present paper, miRNA expression profiles were determined during cardiomyocyte-specific differentiation and maturation of murine embryonic stem (ES) cells. For this purpose a homogeneous cardiomyocyte population was generated from a transgenic murine ES cell line. Two high throughput array platforms (Affymetrix and Febit) were used for miRNA profiling in order to compare the effect of the platforms on miRNA profiling as well as to increase the validity of target miRNA identification. Four time points (i.e. day 0, day 12, day 19 and day 26) were chosen for the miRNA profiling study, which corresponded to different stages during cardiomyocyte-specific differentiation and maturation. Fifty platform and pre-processing method-independent miRNAs were identified as being regulated during the differentiation and maturation processes. The identification of these miRNAs is an important step for characterizing and understanding the events involved in cardiomyocyte-specific differentiation of ES cells and may also highlight candidate target molecules for therapeutic purposes.
Identification of Novel miRNAs and miRNA Dependent Developmental Shifts of Gene Expression in Arabidopsis thaliana  [PDF]
Shuhua Zhan,Lewis Lukens
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0010157
Abstract: microRNAs (miRNAs) are small, endogenous RNAs of 20~25 nucleotides, processed from stem-loop regions of longer RNA precursors. Plant miRNAs act as negative regulators of target mRNAs predominately by slicing target transcripts, and a number of miRNAs play important roles in development. We analyzed a number of published datasets from Arabidopsis thaliana to characterize novel miRNAs, novel miRNA targets, and miRNA-regulated developmental changes in gene expression. These data include microarray profiling data and small RNA (sRNA) deep sequencing data derived from miRNA biogenesis/transport mutants, microarray profiling data of mRNAs in a developmental series, and computational predictions of conserved genomic stem-loop structures. Our conservative analyses identified five novel mature miRNAs and seven miRNA targets, including one novel target gene. Two complementary miRNAs that target distinct mRNAs were encoded by one gene. We found that genes targeted by known miRNAs, and genes up-regulated or down-regulated in miRNA mutant inflorescences, are highly expressed in the wild type inflorescence. In addition, transcripts upregulated within the mutant inflorescences were abundant in wild type leaves and shoot meristems and low in pollen and seed. Downregulated transcripts were abundant in wild type pollen and seed and low in shoot meristems, roots and leaves. Thus, disrupting miRNA function causes the inflorescence transcriptome to resemble the leaf and meristem and to differ from pollen and seed. Applications of our computational approach to other species and the use of more liberal criteria than reported here will further expand the number of identified miRNAs and miRNA targets. Our findings suggest that miRNAs have a global role in promoting vegetative to reproductive transitions in A. thaliana.
Comparison of Microarray Platforms for Measuring Differential MicroRNA Expression in Paired Normal/Cancer Colon Tissues  [PDF]
Maurizio Callari, Matteo Dugo, Valeria Musella, Edoardo Marchesi, Giovanna Chiorino, Maurizia Mello Grand, Marco Alessandro Pierotti, Maria Grazia Daidone, Silvana Canevari, Loris De Cecco
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0045105
Abstract: Background Microarray technology applied to microRNA (miRNA) profiling is a promising tool in many research fields; nevertheless, independent studies characterizing the same pathology have often reported poorly overlapping results. miRNA analysis methods have only recently been systematically compared but only in few cases using clinical samples. Methodology/Principal Findings We investigated the inter-platform reproducibility of four miRNA microarray platforms (Agilent, Exiqon, Illumina, and Miltenyi), comparing nine paired tumor/normal colon tissues. The most concordant and selected discordant miRNAs were further studied by quantitative RT-PCR. Globally, a poor overlap among differentially expressed miRNAs identified by each platform was found. Nevertheless, for eight miRNAs high agreement in differential expression among the four platforms and comparability to qRT-PCR was observed. Furthermore, most of the miRNA sets identified by each platform are coherently enriched in data from the other platforms and the great majority of colon cancer associated miRNA sets derived from the literature were validated in our data, independently from the platform. Computational integration of miRNA and gene expression profiles suggested that anti-correlated predicted target genes of differentially expressed miRNAs are commonly enriched in cancer-related pathways and in genes involved in glycolysis and nutrient transport. Conclusions Technical and analytical challenges in measuring miRNAs still remain and further research is required in order to increase consistency between different microarray-based methodologies. However, a better inter-platform agreement was found by looking at miRNA sets instead of single miRNAs and through a miRNAs – gene expression integration approach.
Performance evaluation of commercial miRNA expression array platforms
Sachin Sah, Matthew N McCall, Deepa Eveleigh, Michael Wilson, Rafael A Irizarry
BMC Research Notes , 2010, DOI: 10.1186/1756-0500-3-80
Abstract: We compared the performance characteristics of four commercial miRNA array technologies and found that all platforms performed well in separate measures of performance.The Ambion and Agilent platforms were more accurate, whereas the Illumina and Exiqon platforms were more specific. Furthermore, the data analysis approach had a large impact on the performance, predominantly by improving precision.MicroRNAs (miRNAs) are endogenous, non-coding transcripts that regulate a diverse range of functions, including development, differentiation, growth, apoptosis and metabolism. These 17-24 nucleotide RNA molecules confer specific recognition of target mRNAs and modulate gene expression by acting in conjunction with a set of effector proteins of the RNA interference pathway [1,2]. Through this interaction, miRNAs negatively regulate expression of specific target mRNAs by inhibiting translation, sequestering transcripts in P-bodies [3], or by accelerating mRNA decay as a consequence of rapid deadenylation[4]. Moreover, miRNAs have recently been proposed to activate translation of mRNAs under certain conditions [5].The relative abundance of miRNAs in cells is thought to be important for miRNAs to exert their regulatory function. For example, titrated expression of both genomic copies of mouse miR-1 is required for normal heart formation and function during embryogenesis [6]. Aberrant miRNA expression contributes to malignancies, tumor progression and metastasis (reviewed in [7]), and miRNA expression profiles can be correlated with disease pathogenesis and prognosis [8,9]. Thus, the performance characteristics of technologies that measure the relative abundance of miRNAs is important for effectively deciphering their functional roles and their potential utility as diagnostic biomarkers.Microarray technology permits simultaneous expression measurements for hundreds of miRNAs. This technology is already widely used and promises to become a standard tool in the near future. However
Differential expression patterns of conserved miRNAs and isomiRs during Atlantic halibut development
Teshome T Bizuayehu, Carlos FC Lanes, Tomasz Furmanek, B?rd O Karlsen, Jorge MO Fernandes, Steinar D Johansen, Igor Babiak
BMC Genomics , 2012, DOI: 10.1186/1471-2164-13-11
Abstract: miRNA profiling using SOLiD deep sequencing technology revealed a total of 199 conserved, one novel antisense, and one miRNA* mature form. Digital expression profiles of selected miRNAs were validated using reverse transcription quantitative PCR. We found developmental transition-specific miRNA expression. Expression of some miRNA* exceeded the guide strand miRNA. We revealed that nucleotide truncations and/or additions at the 3' end of mature miRNAs resulted in size variants showing differential expression patterns during the development in a number of miRNA families. We confirmed the presence of isomiRs by cloning and Sanger sequencing. Also, we found inverse relationship between expression levels of sense/antisense miRNAs during halibut development.Developmental transitions during early development of Atlantic halibut are associated with expression of certain miRNA types. IsomiRs are abundant and often show differential expression during the development.Atlantic halibut, Hippoglossus hippoglossus L., the largest flatfish of Atlantic Ocean, is a species of commercial interest to the aquaculture industry. Halibut's early developmental stages are prolonged and morphologically defined [1,2]. The critical developmental stages, when dramatic changes in signaling, physiology and morphology occur, include: (i) maternal to zygote transition (MZT), when maternally stocked transcripts are degraded and zygote transcripts take control over the development; (ii) organogenesis, when the germ layers are formed; (iii) hatching, when the embryo becomes a free-swimming larva; (iv) first feeding, when active movement, visualization, recognition of prey, and exogenous feeding begin; and (v) metamorphosis, the most dramatic morphological and behavioral change in a flatfish during the transition from a symmetric post-larval to an asymmetric juvenile stage, when migration of one eye towards the other one occurs across the skull [1].MiRNAs are small (18 - 26 nucleotides) non-coding RNAs
miRNA-mRNA Integrated Analysis Reveals Roles for miRNAs in Primary Breast Tumors  [PDF]
Espen Enerly,Israel Steinfeld,Kristine Kleivi,Suvi-Katri Leivonen,Miriam R. Aure,Hege G. Russnes,Jo Anders R?nneberg,Hilde Johnsen,Roy Navon,Einar R?dland,Rami M?kel?,Bj?rn Naume,Merja Per?l?,Olli Kallioniemi,Vessela N. Kristensen,Zohar Yakhini,Anne-Lise B?rresen-Dale
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0016915
Abstract: Few studies have performed expression profiling of both miRNA and mRNA from the same primary breast carcinomas. In this study we present and analyze data derived from expression profiling of 799 miRNAs in 101 primary human breast tumors, along with genome-wide mRNA profiles and extensive clinical information.
Investigation of miRNA Biology by Bioinformatic Tools and Impact of miRNAs in Colorectal Cancer: Regulatory Relationship of c-Myc and p53 with miRNAs
Yaguang Xi,John R. Edwards,Jingfang Ju
Cancer Informatics , 2007,
Abstract: MicroRNAs (miRNAs) are a class of small non-coding RNAs that mediate gene expression at the posttranscriptional and translational levels and have been demonstrated to be involved in diverse biological functions. Mounting evidence in recent years has shown that miRNAs play key roles in tumorigenesis due to abnormal expression of and mutations in miRNAs. High throughput miRNA expression profiling of several major tumor types has identified miRNAs associated with clinical diagnosis and prognosis of cancer treatment. Previously our group has discovered a novel regulatory relationship between tumor suppressor gene p53 with miRNAs expression and a number of miRNA promoters contain putative p53 binding sites. In addition, others have reported that c-myc can mediate a large number of miRNAs expression. In this review, we will emphasize algorithms to identify mRNA targets of miRNAs and the roles of miRNAs in colorectal cancer. In particular, we will discuss a novel regulatory relationship of miRNAs with tumor suppressor p53 and c-myc. miRNAs are becoming promising novel targets and biomarkers for future cancer therapeutic development and clinical molecular diagnosis.
Improvement of tissue preparation for laser capture microdissection: application for cell type-specific miRNA expression profiling in colorectal tumors
Shuyang Wang, Lei Wang, Tengfang Zhu, Xue Gao, Jian Li, Ying Wu, Hongguang Zhu
BMC Genomics , 2010, DOI: 10.1186/1471-2164-11-163
Abstract: We found that the ethanol fixation of tissue sections for 2 hours had the maximum improvement of RNA quality (1.8 fold, p = 0.0014) and quantity (1.5 fold, p = 0.066). Overall, the quality (RNA integrity number, RIN) for the microdissected colorectal tissues was 5.2 ± 1.5 (average ± SD) for normal (n = 43), 5.7 ± 1.1 for adenomas (n = 14) and 7.2 ± 1.2 for carcinomas (n = 44). We then compared miRNA expression profiles of 18 colorectal tissues (6 normal, 6 adenomas and 6 carcinomas) between LCM selected epithelial cells versus stromal cells using Agilent miRNA microarrays. We identified 51 differentially expressed miRNAs (p <= 0.001) between these two cell types. We found that the miRNAs in the epithelial cells could differentiate adenomas from normal and carcinomas. However, the miRNAs in the stromal and mixed cells could not separate adenomas from normal tissues. Finally, we applied quantitative RT-PCR to cross-verify the expression patterns of 7 different miRNAs using 8 LCM-selected epithelial cells and found the excellent correlation of the fold changes between the two platforms (R = 0.996).Our study demonstrates the feasibility and potential power of discovering cell type-specific miRNA biomarkers in complex tissue using combination of LCM with genome-wide miRNA analysis.Molecular profiling of clinical tissue specimens is frequently complicated by their cellular heterogeneity. Laser capture microdissection (LCM) has successfully been used to tackle this problem by isolating pure cell populations from tissue sections [1-3] and the combination of LCM with standard genomic and proteomic methods has revolutionized molecular analysis of complex tissue. It has allowed for the discrimination of genomic changes, differential expressions and subsequent signaling effects for a variety of proteins in diagnostic tissues [4-10]. Despite these advances, the quantity and quality of material recovered after LCM is often still limited for analysis by using whole genomic and pro
A systematic analysis of the skeletal muscle miRNA transcriptome of chicken varieties with divergent skeletal muscle growth identifies novel miRNAs and differentially expressed miRNAs
Tingting Li, Rimao Wu, Yong Zhang, Dahai Zhu
BMC Genomics , 2011, DOI: 10.1186/1471-2164-12-186
Abstract: Some miRNAs have multiple isoforms and several miRNAs* are present at higher levels than their corresponding miRNAs. Thirty three novel and 189 known chicken miRNAs were identified using computational approaches. Subsequent miRNA transcriptome comparisons and real-time PCR validation experiments revealed 17 miRNAs that were differentially expressed between broilers and layers, and a number of targets of these miRNAs have been implicated in myogenesis regulation. Using integrative miRNA target-prediction and network-analysis approaches an interaction network of differentially expressed and muscle-related miRNAs and their putative targets was constructed, and miRNAs that could contribute to the divergent muscle growth of broiler and layer chickens by targeting the ACVR2B gene were identified, which can causes dramatic increases in muscle mass.The present study provides the first transcriptome profiling-based evaluation of miRNA function during skeletal muscle development in chicken. Systematic predictions aided the identification of potential miRNAs and their targets, which could contribute to divergent muscle growth in broiler and layer chickens. Furthermore, these predictions generated information that can be utilized in further research investigating the involvement of interaction networks, containing miRNAs and their targets, in the regulation of muscle development.Embryonic patterning and organogenesis involve coordinated differentiation, migration, proliferation and programmed cell death in metazoans. These complex cellular and developmental processes rely on precise spatiotemporal networks that regulate transcription factors at multiple levels including mRNA transcription and translation, protein stability and degradation. Recently, evidence has demonstrated that microRNAs (miRNAs or miRs) are involved in diverse aspects of biology including developmental regulation and the pathogenesis of human diseases [1-4]. miRNAs are small 19-24 nucleotide (nt) regulatory
Quantitative miRNA Expression Analysis Using Fluidigm Microfluidics Dynamic Arrays
Jin Jang, Vernadette A Simon, Rod M Feddersen, Fariborz Rakhshan, Debra A Schultz, Michael A Zschunke, Wilma L Lingle, Christopher P Kolbert, Jin Jen
BMC Genomics , 2011, DOI: 10.1186/1471-2164-12-144
Abstract: We demonstrated highly correlated Ct values between multiplex and singleplex RT reactions in standard qPCR assays for miRNA expression using total RNA from A549 (R = 0.98; p < 0.0001) and H1299 (R = 0.95; p < 0.0001) lung cancer cell lines. The Ct values generated by the microfluidic technology (Fluidigm 48.48 dynamic array systems) resulted in a left-shift toward lower Ct values compared to those observed by ABI 7900 HT (mean difference, 3.79), suggesting that the microfluidic technology exhibited a greater sensitivity. In addition, we show that as little as 10 ng total RNA can be used to reliably detect all 48 or 96 tested miRNAs using a 96-multiplexing RT reaction in both FFPE and FF samples. Finally, we compared miRNA expression measurements in both FFPE and FF samples by qPCR using the 96.96 dynamic array and Affymetrix microarrays. Fold change comparisons for comparable genes between the two platforms indicated that the overall correlation was R = 0.60. The maximum fold change detected by the Affymetrix microarray was 3.5 compared to 13 by the 96.96 dynamic array.The qPCR-array based microfluidic dynamic array platform can be used in conjunction with multiplexed RT reactions for miRNA gene expression profiling. We showed that this approach is highly reproducible and the results correlate closely with the existing singleplex qPCR platform at a throughput that is 5 to 20 times higher and a sample and reagent usage that was approximately 50-100 times lower than conventional assays. We established optimal conditions for using the Fluidigm microfluidic technology for rapid, cost effective, and customizable arrays for miRNA expression profiling and validation.MicroRNAs (miRNAs) are short, single-stranded, noncoding RNAs that regulate gene expression by interacting with or inhibiting mRNA in both plants and animals [1-3]. To date, more than 800 human miRNAs have been identified and the total number is still increasing [4]. It is estimated that about two thirds of all
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