Sequencing Of Small RNA Libraries Research Articles

Analysis of microRNA transcriptome by deep sequencing of small RNA libraries of peripheral blood

BackgroundMicroRNAs are a class of small non-coding RNAs that regulate mRNA expression at the post - transcriptional level and thereby many fundamental biological processes. A number of methods, such as multiplex polymerase chain reaction, microarrays have been developed for profiling levels of known miRNAs. These methods lack the ability to identify novel miRNAs and accurately determine expression at a range of concentrations. Deep or massively parallel sequencing methods are providing suitable platforms for genome wide transcriptome analysis and have the ability to identify novel transcripts.ResultsThe results of analysis of small RNA sequences obtained by Solexa technology of normal peripheral blood mononuclear cells, tumor cell lines K562 and HL60 are presented. In general K562 cells displayed overall low level of miRNA population and also low levels of DICER. Some of the highly expressed miRNAs in the leukocytes include several members of the let-7 family, miR-21, 103, 185, 191 and 320a. Comparison of the miRNA profiles of normal versus K562 or HL60 cells revealed a specific set of differentially expressed molecules. Correlation of the miRNA with that of mRNA expression profiles, obtained by microarray, revealed a set of target genes showing inverse correlation with miRNA levels. Relative expression levels of individual miRNAs belonging to a cluster were found to be highly variable. Our computational pipeline also predicted a number of novel miRNAs. Some of the predictions were validated by Real-time RT-PCR and or RNase protection assay. Organization of some of the novel miRNAs in human genome suggests that these may also be part of existing clusters or form new clusters.ConclusionsWe conclude that about 904 miRNAs are expressed in human leukocytes. Out of these 370 are novel miRNAs. We have identified miRNAs that are differentially regulated in normal PBMC with respect to cancer cells, K562 and HL60. Our results suggest that post - transcriptional processes may play a significant role in regulating levels of miRNAs in tumor cells. The study also provides a customized automated computation pipeline for miRNA profiling and identification of novel miRNAs; even those that are missed out by other existing pipelines. The Computational Pipeline is available at the website: http://mirna.jnu.ac.in/deep_sequencing/deep_sequencing.html

Abstract 1987: RNA-seq of the bronchial airway transcriptome identifies novel gene and microRNA expression changes associated with smoking and lung cancer

Abstract Prior studies have shown that cigarette smoke creates a field of molecular injury in the airway epithelium. Using microarrays, our group has previously identified gene and microRNA expression differences in bronchial airway epithelial cells that are associated with smoking and lung cancer. We hypothesize that RNA-seq of the airway transcriptome will enhance our understanding of the response to tobacco smoke exposure and lung cancer pathogenesis by identifying mRNA splice variants, non-coding RNA, and novel RNA not interrogated by microarrays. We profiled pooled bronchial airway epithelial cell brushings (n=3 patients/pool) obtained during bronchoscopy from healthy never and current smoker volunteers and smokers with and without lung cancer undergoing surgery for suspicion of lung cancer. The high MW fraction (>200 bp) was amplified using a combination of oligo d(T) and random hexamers (NuGEN, San Carlos, CA) followed by library preparation with Illumina's mRNA Seq Library Prep Kit and sequencing using Illumina's Genome Analyzer generating ∼27-30 million 36 bp reads/sample. The low MW fraction (15-40 bp) was processed using ABI's Small RNA Library Sequencing Kit and sequenced using ABI's SOLiD system generating ∼50-90 million 35 bp reads/sample. The high MW reads were aligned to rRNA (5-13%), the human genome (33-40%), and to computationally generated splice junctions (1%) using BowTie. Differential gene and isoform expression was evaluated using Cufflinks and the R package Genominator. Low MW reads were aligned using both RNA2MAP and Geospiza to a filter containing rRNA, tRNA, and repeats (4-14%), to miRBase (4-10%), and the human genome minus miRBase sequences (4-8%). Expression values were calculated for microRNAs. We compared Affymetrix Exon 1.0 ST, HGU133A2, and Invitrogen miRNA microarrays to RNA-seq and found strong correlation for genes/miRNAs interrogated by microarrays, with most smoking- and cancer related changes in transcript expression being identified by sequencing and not by arrays. Genes found differentially expressed only by sequencing were validated by RT-PCR. RNA-seq also reveals differentially expressed isoforms, potential novel miRNAs, and several isoforms of known miRNAs which are being further investigated. RNA-seq provides a comprehensive and high-resolution view of the airway transcriptome and will provide insights into the molecular field of injury induced by smoking and the pathogenesis of smoking-related lung disease. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1987.

Deep sequencing of small RNA libraries reveals dynamic regulation of conserved and novel microRNAs and microRNA-stars during silkworm development

BackgroundIn eukaryotes, microRNAs (miRNAs) have emerged as critical regulators of gene expression. The Silkworm (Bombyx mori L.) is one of the most suitable lepidopteran insects for studying the molecular aspects of metamorphosis because of its large size, availability of mutants and genome sequence. Besides, this insect also has been amply studied from a physiological and biochemical perspective. Deep sequencing of small RNAs isolated from different stages of silkworm is a powerful tool not only for measuring the changes in miRNA profile but also for discovering novel miRNAs.ResultsWe generated small RNA libraries from feeding larvae, spinning larvae, pupae and adults of B. mori and obtained ~2.5 million reads of 18-30 nt. Sequence analysis identified 14 novel and 101 conserved miRNAs. Most novel miRNAs are preferentially expressed in pupae, whereas more than 95% of the conserved miRNAs are dynamically regulated during different developmental stages. Remarkably, the miRNA-star (miR*) of four miRNAs are expressed at much higher levels than their corresponding miRNAs, and their expression profiles are distinct from their corresponding miRNA profiles during different developmental stages. Additionally, we detected two antisense miRNA loci (miR-263-S and miR-263-AS; miR-306-S and miR-306-AS) that are expressed in sense and antisense directions. Interestingly, miR-263 and miR-306 are preferentially and abundantly expressed in pupae and adults, respectively.ConclusionsWe identified 101 homologs of conserved miRNAs, 14 species-specific and two antisense miRNAs in the silkworm. Our results provided deeper insights into changes in conserved and novel miRNA and miRNA* accumulation during development.

Functional Shifts in Insect microRNA Evolution

MicroRNAs (miRNAs) are short endogenous RNA molecules that regulate gene expression at the posttranscriptional level and have been shown to play critical roles during animal development. The identification and comparison of miRNAs in metazoan species are therefore paramount for our understanding of the evolution of body plans. We have characterized 203 miRNAs from the red flour beetle Tribolium castaneum by deep sequencing of small RNA libraries. We can conclude, from a single study, that the Tribolium miRNA set is at least 15% larger than that in the model insect Drosophila melanogaster (despite tens of high-throughput sequencing experiments in the latter). The rate of birth and death of miRNAs is high in insects. Only one-third of the Tribolium miRNA sequences are conserved in D. melanogaster, and at least 18 Tribolium miRNAs are conserved in vertebrates but lost in Drosophila. More than one-fifth of miRNAs that are conserved between Tribolium and Drosophila exhibit changes in the transcription, genomic organization, and processing patterns that lead to predicted functional shifts. For example, 13% of conserved miRNAs exhibit seed shifting, and we describe arm-switching events in 11% of orthologous pairs. These shifts fundamentally change the predicted targets and therefore function of orthologous miRNAs. In general, Tribolium miRNAs are more representative of the insect ancestor than Drosophila miRNAs and are more conserved in vertebrates.

Mir-886-3p Contributes to the Regulation of the hematopoietic microenvironment by down-Regulating SDF-1α (CXCL12).

Abstract 561Two functionally distinct stromal cell lines were isolated from a primary long term culture (LTC) established from aspirated human marrow. Designated HS5 and HS27a, the lines were immortalized and extensively characterized including expression profiles for both messenger (mRNA) and micro-RNA (miRNA, a recently described class of small non-coding RNAs that regulate gene expression by binding to target mRNAs to prevent their translation). HS5 was found to secrete growth factors that stimulate proliferation and differentiation of hematopoietic progenitors (G-CSF, IL-6, IL-1α and IL1β), whereas HS27a expresses activities associated with the stem cell niche (SDF-1αa, angiopoietin-1 etc). In keeping with this HS5 conditioned media stimulated proliferation and differentiation of isolated CD34+ cells whereas HS27a supported CD34+ cells in an undifferentiated state. When cultured together to better mimic in vivo cell-cell interactions, the gene expression of HS27a and HS5 combined differed from the expected sum of the two parts, exemplified by the 5-fold down regulation of SDF-1α.Comparisons of miRNA expression profiles of HS5 and HS27a determined that mir-886-3p, (previously described by deep sequencing of small RNA libraries) was expressed > 40 fold in HS5 compared to HS27a, this was then confirmed by quantitative RT-PCR. Given the abundance of mir-886-3p and the possibility that it could be secreted by HS5 and taken up by cells in contact with HS5, we tested its effect on gene expression in HS27a. Transcript levels of genes associated with the stem cell niche (Jagged1, BMP4, Angiopoietin-1, SDF-1α, VCAM-1 and N-Cadherin) were determined by quantitative RT-PCR after direct transfection of mir-886-3p precursors into HS27a cells and compared to appropriate controls. Results show SDF-1α mRNA expression was down-regulated by as much as 8 fold 3 days after transfection. Levels of secreted SDF-1α in culture media, as determined by ELISA, were also decreased. Since SDF-1α is a chemokine known to be critical for the homing of hematopoietic stem and progenitor cells to their niche, the functional significance of the SDF-1α down-regulation by mir-886-3p was confirmed by decreased chemotaxis of T-lymphocytic cells (Jurkat) following miRNA transfection of stromal cells. To determine if mir-886-3p directly effects the SDF-1α transcript, the 1.5 kbp 3'untranslated region (UTR) of SDF-1α gene was cloned downstream of the luciferase gene, and co-transfected with mir-886-3p into HS27a cells. Results showed the luciferase activity was down-regulated greater than 50% in the presence of mir-886-3p, suggesting a direct effect on the SDF-1 α transcript.Given the concern over the relevance of immortalized cell lines we investigated Mir-886-3p expression in primary marrow stromal cells at early passage sorted on the basis of +/- expression of CD146. (CD146 or MCAM has been reported to define a population that supports the hematopoietic stem/ precursor cell niche and is expressed by HS27a and not HS5 cells). Results indicated that the CD146+ stromal cells had significantly lower expression of mir-886-3p when compared to CD146- cells.In summary, these data suggest a role for miRNA in modulating the expression of gene products that are associated with the hematopoietic stem cell niche. Disclosures:No relevant conflicts of interest to declare.

Hypoxia-responsive microRNAs and trans-acting small interfering RNAs in Arabidopsis.

Low-oxygen (hypoxia) stress associated with natural phenomena such as waterlogging, results in widespread transcriptome changes and a metabolic switch from aerobic respiration to anaerobic fermentation. High-throughput sequencing of small RNA libraries obtained from hypoxia-treated and control root tissue identified a total of 65 unique microRNA (miRNA) sequences from 46 families, and 14 trans-acting small interfering RNA (tasiRNA) from three families. Hypoxia resulted in changes to the abundance of 46 miRNAs from 19 families, and all three tasiRNA families. Chemical inhibition of mitochondrial respiration caused similar changes in expression in a majority of the hypoxia-responsive small RNAs analysed. Our data indicate that miRNAs and tasiRNAs play a role in gene regulation and possibly developmental responses to hypoxia, and that a major signal for these responses is likely to be dependent on mitochondrial function.

Systematic and single cell analysis of Xenopus Piwi-interacting RNAs and Xiwi

Piwi proteins and Piwi-interacting RNAs (piRNAs) are essential for germ cell development, but analysis of the molecular mechanisms of these ribonucleoproteins remains challenging in most animal germ cells. To address this challenge, we systematically characterized Xiwi, a Xenopus Piwi homologue, and piRNAs from Xenopus eggs and oocytes. We used the large size of Xenopus eggs to analyze small RNAs at the single cell level, and find abundant piRNAs and large piRNA clusters in the Xenopus tropicalis genome, some of which resemble the Drosophila piRNA-generating flamenco locus. Although most piRNA clusters are expressed simultaneously in an egg, individual frogs show distinct profiles of cluster expression. Xiwi is associated with microtubules and the meiotic spindle, and is localized to the germ plasm--a cytoplasmic determinant of germ cell formation. Xiwi associates with translational regulators in an RNA-dependent manner, but Xenopus tudor interacts with Xiwi independently of RNA. Our study adds insight to piRNA transcription regulation by showing that individual animals can have differential piRNA expression profiles. We suggest that in addition to regulating transposable elements, Xiwi may function in specifying RNA localization in vertebrate oocytes.

Human Villous Trophoblasts Express and Secrete Placenta-Specific MicroRNAs into Maternal Circulation via Exosomes1

In this study, we performed small RNA library sequencing using human placental tissues to identify placenta-specific miRNAs. We also tested the hypothesis that human chorionic villi could secrete miRNAs extracellularly via exosomes, which in turn enter into maternal circulation. By small RNA library sequencing, most placenta-specific miRNAs (e.g., MIR517A) were linked to a miRNA cluster on chromosome 19. The miRNA cluster genes were differentially expressed in placental development. Subsequent validation by real-time PCR and in situ hybridization revealed that villous trophoblasts express placenta-specific miRNAs. The analysis of small RNA libraries from the blood plasma showed that the placenta-specific miRNAs are abundant in the plasma of pregnant women. By real-time PCR, we confirmed the rapid clearance of the placenta-specific miRNAs from the plasma after delivery, indicating that such miRNAs enter into maternal circulation. By using the trophoblast cell line BeWo in culture, we demonstrated that miRNAs are indeed extracellularly released via exosomes. Taken together, our findings suggest that miRNAs are exported from the human placental syncytiotrophoblast into maternal circulation, where they could target maternal tissues. Finally, to address the biological functions of placenta-specific miRNAs, we performed a proteome analysis of BeWo cells transfected with MIR517A. Bioinformatic analysis suggests that this miRNA is possibly involved in tumor necrosis factor-mediated signaling. Our data provide important insights into miRNA biology of the human placenta.

A distinct class of small RNAs arises from pre-miRNA–proximal regions in a simple chordate

MicroRNAs (miRNAs) have been implicated in various cellular processes. They are thought to function primarily as inhibitors of gene activity by attenuating translation or promoting mRNA degradation. A typical miRNA gene produces a predominant approximately 21-nucleotide (nt) RNA (the miRNA) along with a less abundant miRNA(*) product. We sought to identify miRNAs from the simple chordate Ciona intestinalis through comprehensive sequencing of small RNA libraries created from different developmental stages. Unexpectedly, half of the identified miRNA loci encode up to four distinct, stable small RNAs. The additional RNAs, miRNA-offset RNAs (moRs), are generated from sequences immediately adjacent to the predicted approximately 60-nt pre-miRNA. moRs seem to be produced by RNAse III-like processing, are approximately 20 nt long and, like miRNAs, are observed at specific developmental stages. We present evidence suggesting that the biogenesis of moRs results from an intrinsic property of the miRNA processing machinery in C. intestinalis.

The deep evolution of metazoan microRNAs

microRNAs (miRNAs) are approximately 22-nucleotide noncoding RNA regulatory genes that are key players in cellular differentiation and homeostasis. They might also play important roles in shaping metazoan macroevolution. Previous studies have shown that miRNAs are continuously being added to metazoan genomes through time, and, once integrated into gene regulatory networks, show only rare mutations within the primary sequence of the mature gene product and are only rarely secondarily lost. However, because the conclusions from these studies were largely based on phylogenetic conservation of miRNAs between model systems like Drosophila and the taxon of interest, it was unclear if these trends would describe most miRNAs in most metazoan taxa. Here, we describe the shared complement of miRNAs among 18 animal species using a combination of 454 sequencing of small RNA libraries with genomic searches. We show that the evolutionary trends elucidated from the model systems are generally true for all miRNA families and metazoan taxa explored: the continuous addition of miRNA families with only rare substitutions to the mature sequence, and only rare instances of secondary loss. Despite this conservation, we document evolutionary stable shifts to the determination of position 1 of the mature sequence, a phenomenon we call seed shifting, as well as the ability to post-transcriptionally edit the 5' end of the mature read, changing the identity of the seed sequence and possibly the repertoire of downstream targets. Finally, we describe a novel type of miRNA in demosponges that, although shows a different pre-miRNA structure, still shows remarkable conservation of the mature sequence in the two sponge species analyzed. We propose that miRNAs might be excellent phylogenetic markers, and suggest that the advent of morphological complexity might have its roots in miRNA innovation.

MicroRNA (miRNA) cloning analysis reveals sex differences in miRNA expression profiles between adult mouse testis and ovary

MicroRNAs (miRNAs) are endogenous non-coding small RNAs that can regulate the expression of complementary mRNA targets. Identifying tissue-specific miRNAs is the first step toward understanding the biological functions of miRNAs, which include the regulation of tissue differentiation and the maintenance of tissue identity. In this study, we performed small RNA library sequencing in adult mouse testis and ovary to reveal their characteristic organ- and gender-specific profiles and to elucidate the characteristics of the miRNAs expressed in the reproductive system. We obtained 10,852 and 11 744 small RNA clones from mouse testis and ovary respectively (greater than 10,000 clones per organ), which included 6630 (159 genes) and 10,192 (154 genes) known miRNAs. A high level of efficiency of miRNA library sequencing was achieved: 61% (6630 miRNA clones/10,852 small RNA clones) and 87% (10,192/11,744) for adult mouse testis and ovary respectively. We obtained characteristic miRNA signatures in testis and ovary; 55 miRNAs were detected highly, exclusively, or predominantly in adult mouse testis and ovary, and discovered two novel miRNAs. Male-biased expression of miRNAs occurred on the X-chromosome. Our data provide important information on sex differences in miRNA expression that should facilitate studies of the reproductive organ-specific roles of miRNAs.

MicroRNA Discovery and Profiling in Human Embryonic Stem Cells by Deep Sequencing of Small RNA Libraries

We used massively parallel pyrosequencing to discover and characterize microRNAs (miRNAs) expressed in human embryonic stem cells (hESC). Sequencing of small RNA cDNA libraries derived from undifferentiated hESC and from isogenic differentiating cultures yielded a total of 425,505 high-quality sequence reads. A custom data analysis pipeline delineated expression profiles for 191 previously annotated miRNAs, 13 novel miRNAs, and 56 candidate miRNAs. Further characterization of a subset of the novel miRNAs in Dicer-knockdown hESC demonstrated Dicer-dependent expression, providing additional validation of our results. A set of 14 miRNAs (9 known and 5 novel) was noted to be expressed in undifferentiated hESC and then strongly downregulated with differentiation. Functional annotation analysis of predicted targets of these miRNAs and comparison with a null model using non-hESC-expressed miRNAs identified statistically enriched functional categories, including chromatin remodeling and lineage-specific differentiation annotations. Finally, integration of our data with genome-wide chromatin immunoprecipitation data on OCT4, SOX2, and NANOG binding sites implicates these transcription factors in the regulation of nine of the novel/candidate miRNAs identified here. Comparison of our results with those of recent deep sequencing studies in mouse and human ESC shows that most of the novel/candidate miRNAs found here were not identified in the other studies. The data indicate that hESC express a larger complement of miRNAs than previously appreciated, and they provide a resource for additional studies of miRNA regulation of hESC physiology. Disclosure of potential conflicts of interest is found at the end of this article.

Functional characterization of endogenous siRNA target genes in Caenorhabditis elegans

BackgroundSmall interfering RNA (siRNA) molecules mediate sequence specific silencing in RNA interference (RNAi), a gene regulatory phenomenon observed in almost all organisms. Large scale sequencing of small RNA libraries obtained from C. elegans has revealed that a broad spectrum of siRNAs is endogenously transcribed from genomic sequences. The biological role and molecular diversity of C. elegans endogenous siRNA (endo-siRNA) molecules, nonetheless, remain poorly understood. In order to gain insight into their biological function, we annotated two large libraries of endo-siRNA sequences, identified their cognate targets, and performed gene ontology analysis to identify enriched functional categories.ResultsSystematic trends in categorization of target genes according to the specific length of siRNA sequences were observed: 18- to 22-mer siRNAs were associated with genes required for embryonic development; 23-mers were associated uniquely with post-embryonic development; 24–26-mers were associated with phosphorus metabolism or protein modification. Moreover, we observe that some argonaute related genes associate with siRNAs with multiple reads. Sequence frequency graphs suggest that different lengths of siRNAs share similarities in overall sequence structure: the 5' end begins with G, while the body predominates with U and C.ConclusionThese results suggest that the lengths of endogenous siRNA molecules are consequential to their biological functions since the gene ontology categories for their cognate mRNA targets vary depending upon their lengths.

Identification of novel and candidate miRNAs in rice by high throughput sequencing

BackgroundSmall RNA-guided gene silencing at the transcriptional and post-transcriptional levels has emerged as an important mode of gene regulation in plants and animals. Thus far, conventional sequencing of small RNA libraries from rice led to the identification of most of the conserved miRNAs. Deep sequencing of small RNA libraries is an effective approach to uncover rare and lineage- and/or species-specific microRNAs (miRNAs) in any organism.ResultsIn order to identify new miRNAs and possibly abiotic-stress regulated small RNAs in rice, three small RNA libraries were constructed from control rice seedlings and seedlings exposed to drought or salt stress, and then subjected to pyrosequencing. A total of 58,781, 43,003 and 80,990 unique genome-matching small RNAs were obtained from the control, drought and salt stress libraries, respectively. Sequence analysis confirmed the expression of most of the conserved miRNAs in rice. Importantly, 23 new miRNAs mostly each derived from a unique locus in rice genome were identified. Six of the new miRNAs are conserved in other monocots. Additionally, we identified 40 candidate miRNAs. Allowing not more than 3 mis-matches between a miRNA and its target mRNA, we predicted 20 targets for 9 of the new miRNAs.ConclusionDeep sequencing proved to be an effective strategy that allowed the discovery of 23 low-abundance new miRNAs and 40 candidate miRNAs in rice.

MicroRNA Expression Profiling Characterize Diffuse Large B-Cell Lymphomas and Follicular Lymphomas.

A new and powerful tool for classification and understanding the biology of human lymphomas is microRNA (miRNA) expression profiling. MiRNAs comprise a class of approximately 1000 RNA species, which are thought to directly regulate the expression of ∼30% of the transcripts of the human genome on a posttranscriptional level. An important regulatory role of miRNAs has been implicated in biological processes such as cell proliferation and differentiation, fat metabolism, insulin secretion and hematopoiesis. Recent studies indicate that miRNAs are mechanistically involved in the development of various human malignancies and therefore represent a promising new class of biomarkers. We evaluated the miRNA expression profiles of 58 Diffuse Large B-Cell Lymphomas (DLBCL), 46 Follicular Lymphomas (FL) and 7 lymph nodes with chronic lymphadenitis. A quantitative PCR-based method was used to determine the expression levels of 157 miRNA species. The analysis was performed exclusively on FFPE tissues, which are traditionally inapt to molecular analysis. It was possible to clearly distinguish malignant and non-malignant tissues by miRNA expression patterns. We found 34 differentially expressed miRNAs by comparing DLBCL and lymph nodes, 25 miRNAs by comparing FL and lymph nodes and 55 by comparing DLBCL and FL. It was noticeable that the miRNA expression profiles of FL and lymph nodes are more similar compared to miRNA expression profiles of DLBCL. We were also interested to find out whether it is possible to classify the DLBCL cases in germinal center-like DLBCL (GCB-DLBCL) and Non-germinal center-like DLBCL (Non-GCB-DLBCL) based on miRNA expression patterns. We used the immunohistochemical classification, published by Hans et al., to identify both subgroups (Hans CP, Weisenburger DD et al. Confirmation of the molecular classification of diffuse large B-cell lymphoma by immunohistochemistry using a tissue microarray. Blood . 2004; 103:275–282). Comparing miRNA expression profiles of GCB and Non-GCB cases of DLBCL, 8 miRNAs were found to be differentially expressed using the partial least squares discriminant analysis (PLS-DA). However, using a cross-validation approach it was not possible to predict the GCB status on the basis of miRNA expression patterns. Similar to Landgraf et al. we found a reduced miR-150 expression level in DLBCL in comparison to other closely related germinal center malignancies (Landgraf P, Ruscu M et al. A mammalian microRNA expression atlas based on small RNA library sequencing. Cell . 2007; 129:1401–1414). To determine the function of miR-150, Ramos cells were transfected with anti-miR-150 and an analysis of putative target proteins was carried out. It was possible to show a regulation of transcription factor v-myb/c-myc, which plays an important role in the control of proliferation and differentiation of hematopoietic progenitor cells, by miR-150. Moreover, miRNA profiles of DLBCL from a randomized trail will be compared to survival data in the future.

A Mammalian microRNA Expression Atlas Based on Small RNA Library Sequencing

MicroRNAs (miRNAs) are small noncoding regulatory RNAs that reduce stability and/or translation of fully or partially sequence-complementary target mRNAs. In order to identify miRNAs and to assess their expression patterns, we sequenced over 250 small RNA libraries from 26 different organ systems and cell types of human and rodents that were enriched in neuronal as well as normal and malignant hematopoietic cells and tissues. We present expression profiles derived from clone count data and provide computational tools for their analysis. Unexpectedly, a relatively small set of miRNAs, many of which are ubiquitously expressed, account for most of the differences in miRNA profiles between cell lineages and tissues. This broad survey also provides detailed and accurate information about mature sequences, precursors, genome locations, maturation processes, inferred transcriptional units, and conservation patterns. We also propose a subclassification scheme for miRNAs for assisting future experimental and computational functional analyses.

Assessing the risk of hypoxia in flight: the need for more rational guidelines

<h3>ABSTRACT</h3> Plant microRNAs (miRNAs) are short, non-coding RNA molecules that restrict gene expression via post-transcriptional regulation and function in several essential pathways including development, growth, and stress responses. Accurately identifying miRNAs in populations of small RNA (sRNA) sequencing libraries is a computationally intensive process which has resulted in the misidentification of inaccurately annotated miRNA sequences. In recent years, criteria for miRNA annotation have been refined to reduce these misannotations. Here, we describe <i>miRador</i>, a novel miRNA identification tool that utilizes the most up-to-date, community-established criteria for accurate identification of miRNAs in plants. We combine target prediction and Parallel Analysis of RNA Ends (PARE) data to assess the precision of the miRNAs identified by <i>miRador</i>. We compare <i>miRador</i> to other commonly used miRNA prediction tools and we find that <i>miRador</i> is at least as precise as other prediction tools while being significantly faster than other tools.