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MicroRNA sequencing

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344: 208: 35:, also called miRNAs. miRNA-seq differs from other forms of RNA-seq in that input material is often enriched for small RNAs. miRNA-seq allows researchers to examine tissue-specific expression patterns, disease associations, and isoforms of miRNAs, and to discover previously uncharacterized miRNAs. Evidence that dysregulated miRNAs play a role in diseases such as 160:. However, this method is exhaustive in terms of time and resources, as each clone has to be individually amplified and prepared for sequencing. This method also inadvertently favors miRNAs that are highly expressed. Next-generation sequencing eliminates the need for sequence specific hybridization probes required in 4243:
Linsen, Sam E V; de Wit, Elzo; Janssens, Georges; Heater, Sheila; Chapman, Laura; Parkin, Rachael K; Fritz, Brian; Wyman, Stacia K; de Bruijn, Ewart; Voest, Emile E; Kuersten, Scott; Tewari, Muneesh; Cuppen, Edwin (2009). "Limitations and possibilities of small RNA digital gene expression profiling".
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Cloonan, Nicole; Wani, Shivangi; Xu, Qinying; Gu, Jian; Lea, Kristi; Heater, Sheila; Barbacioru, Catalin; Steptoe, Anita L; Martin, Hilary C; Nourbakhsh, Ehsan; Krishnan, Keerthana; Gardiner, Brooke; Wang, Xiaohui; Nones, Katia; Steen, Jason A; Matigan, Nick; Wood, David L; Kassahn, Karin S; Waddell,
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Keller, Andreas; Backes, Christina; Leidinger, Petra; Kefer, Nathalie; Boisguerin, Valesca; Barbacioru, Catalin; Vogel, Britta; Matzas, Mark; Huwer, Hanno; Katus, Hugo A.; Stähler, Cord; Meder, Benjamin; Meese, Eckart (2011). "Next-generation sequencing identifies novel microRNAs in peripheral blood
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Many miRNAs function to direct cleavage of their mRNA targets; this is particularly true in plants, and thus high-throughput sequencing methods have been developed to take advantage of this property of miRNAs by sequencing the uncapped 3' ends of cleaved or degraded mRNAs. These methods are known as
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After the abundances of miRNAs are quantified for each sample, their expression levels can be compared between samples. One would then be able to identify miRNA that are preferentially expressed that particular time points, or in particular tissues or disease states. After normalizing for the number
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Isolated RNA is run on a denaturing polyacrylamide gel. An imaging method such as radioactive 5’-32P-labeled oligonucleotides along with a size ladder is used to identify a section of the gel containing RNA of the appropriate size, reducing the amount of material ultimately sequenced. This step
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Marcucci, Guido; Radmacher, Michael D.; Maharry, Kati; Mrózek, Krzysztof; Ruppert, Amy S.; Paschka, Peter; Vukosavljevic, Tamara; Whitman, Susan P.; Baldus, Claudia D.; Langer, Christian; Liu, Chang-Gong; Carroll, Andrew J.; Powell, Bayard L.; Garzon, Ramiro; Croce, Carlo M.; Kolitz, Jonathan E.;
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The disadvantages of using miRNA-seq over other methods of miRNA profiling are that it is more expensive, generally requires a larger amount of total RNA, involves extensive amplification, and is more time-consuming than microarray and qPCR methods. As well, miRNA-seq library preparation methods
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miRNAs may be preferentially expressed in certain cell types, tissues, stages of development, or in particular disease states such as cancer. Since deep sequencing (miRNA-seq) generates millions of reads from a given sample, it allows us to profile miRNAs; whether it may be by quantifying their
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Ueda, Tetsuya; Volinia, Stefano; Okumura, Hiroshi; Shimizu, Masayoshi; Taccioli, Cristian; Rossi, Simona; Alder, Hansjuerg; Liu, Chang-gong; Oue, Naohide; Yasui, Wataru; Yoshida, Kazuhiro; Sasaki, Hiroki; Nomura, Sachiyo; Seto, Yasuyuki; Kaminishi, Michio; Calin, George A; Croce, Carlo M (2010).
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Calin, George Adrian; Ferracin, Manuela; Cimmino, Amelia; Di Leva, Gianpiero; Shimizu, Masayoshi; Wojcik, Sylwia E.; Iorio, Marilena V.; Visone, Rosa; Sever, Nurettin Ilfer; Fabbri, Muller; Iuliano, Rodolfo; Palumbo, Tiziana; Pichiorri, Flavia; Roldo, Claudia; Garzon, Ramiro; Sevignani, Cinzia;
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sequencing platform. This study demonstrated the potential of novel, high-throughput sequencing technologies for the study of small RNAs, and it showed that genomes generate large numbers of small RNAs with plants as particularly rich sources of small RNAs. Later studies used other sequencing
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Jima, D. D.; Zhang, J.; Jacobs, C.; Richards, K. L.; Dunphy, C. H.; Choi, W. W. L.; Yan Au, W.; Srivastava, G.; Czader, M. B.; Rizzieri, D. A.; Lagoo, A. S.; Lugar, P. L.; Mann, K. P.; Flowers, C. R.; Bernal-Mizrachi, L.; Naresh, K. N.; Evens, A. M.; Gordon, L. I.; Luftig, M.; Friedman, D. R.;
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amplification. An adenylated single strand DNA 3’adaptor followed by a 5’adaptor is ligated to the small RNAs using a ligating enzyme such as T4 RNA ligase2. The adaptors are also designed to capture small RNAs with a 5’ phosphate group, characteristic microRNAs, rather than RNA degradation
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expression. In combination with the development of high-throughput profiling methods, miRNAs have been identified as biomarkers for cancer classification, response to therapy, and prognosis. Additionally, because miRNAs regulate gene expression they can also reveal perturbations in important
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Identifying a miRNA's mRNA targets will provide an understanding of the genes or networks of genes whose expression they regulate. Public databases provide predictions of miRNA targets. But to better distinguish true positive predictions from false positive predictions, miRNA-seq data can be
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has positioned miRNA-seq to potentially become an important tool in the future for diagnostics and prognostics as costs continue to decrease. Like other miRNA profiling technologies, miRNA-Seq has both advantages (sequence-independence, coverage) and disadvantages (high cost, infrastructure
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miRNA-seq has revealed novel miRNAs that were previously eluded in traditional miRNA profiling methods. Examples of such findings are in embryonic stem cells, chicken embryos, acute lymphoblastic leukaemia, diffuse large b-cell lymphoma and b-cells, acute myeloid leukemia, and lung cancer.
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Iorio, M. V.; Ferracin, M.; Liu, C.-G.; Veronese, A.; Spizzo, R.; Sabbioni, S.; Magri, E.; Pedriali, M.; Fabbri, M.; Campiglio, M.; Menard, S.; Palazzo, J. P.; Rosenberg, A.; Musiani, P.; Volinia, S.; Nenci, I.; Calin, G. A.; Querzoli, P.; Negrini, M.; Croce, C. M. (2005).
259:) reagent. A starting quantity of 50-100 μg total RNA, 1 g of tissue typically yields 1 mg of total RNA, is usually required for gel purification and size selection. Quality control of the RNA is also measured, for example running an RNA chip on Caliper LabChipGX ( 361:) Note that given that the average length of sequence reads are longer than the average miRNA (17-25 nt), the 3’ and 5’ ends of the miRNA should be found on the same read. There are several miRNA abundance quantification algorithms. Their general steps are as follows: 4026:
Garzon, R.; Garofalo, M.; Martelli, M. P.; Briesewitz, R.; Wang, L.; Fernandez-Cymering, C.; Volinia, S.; Liu, C.-G.; Schnittger, S.; Haferlach, T.; Liso, A.; Diverio, D.; Mancini, M.; Meloni, G.; Foa, R.; Martelli, M. F.; Mecucci, C.; Croce, C. M.; Falini, B. (2008).
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Fridman, Eddie; Dotan, Zohar; Barshack, Iris; David, Miriam Ben; Dov, Avital; Tabak, Sarit; Zion, Orit; Benjamin, Sima; Benjamin, Hila; Kuker, Hagit; Avivi, Camila; Rosenblatt, Kinneret; Polak-Charcon, Sylvie; Ramon, Jacob; Rosenfeld, Nitzan; Spector, Yael (2010).
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German MA, Pillay M, Jeong DH, Hetawal A, Luo S, Janardhanan P, Kannan V, Rymarquis LA, Nobuta K, German R, De Paoli E, Lu C, Schroth G, Meyers BC, Green PJ (2008). "Global identification of microRNA-target RNA pairs by parallel analysis of RNA ends".
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analysis as well as laborious cloning methods required in the Sanger sequencing method. Additionally, next-generation sequencing platforms in the miRNA-SEQ method facilitate the sequencing of large pools of small RNAs in a single sequencing run.
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Central to miRNA-seq data analysis is the ability to 1) obtain miRNA abundance levels from sequence reads, 2) discover novel miRNAs and then be able to 3) determine the differentially expressed miRNA and their 4) associated mRNA gene targets.
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has-miR-128, has-miR-129-3p, has-miR-152, has-miR-155, has-miR-185, has-miR-193a-5p, has-miR-196b, has-miR-199b-3p, has-miR-20b, has-miR-23a, has-miR-27a, has-miR-28-5p, has-miR-301a, has-miR-331-3p, has-miR-365, has-miR-625, has-miR-9
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Another advantage of miRNA-seq is that it allows the discovery of novel miRNAs that may have eluded traditional screening and profiling methods. There are several novel miRNA discovery algorithms. Their general steps are as follows:
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Lebanony, D.; Benjamin, H.; Gilad, S.; Ezagouri, M.; Dov, A.; Ashkenazi, K.; Gefen, N.; Izraeli, S.; Rechavi, G.; Pass, H.; Nonaka, D.; Li, J.; Spector, Y.; Rosenfeld, N.; Chajut, A.; Cohen, D.; Aharonov, R.; Mansukhani, M. (2009).
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seem to have systematic preferential representation of the miRNA complement, and this prevents accurate determination of miRNA abundance. At the same time, the approach is hybridization independent and therefore does not require
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Sequence library construction can be performed using a variety of different kits depending on the high-throughput sequencing platform being employed. However, there are several common steps for small RNA sequencing preparation.
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Gutiérrez, N C; Sarasquete, M E; Misiewicz-Krzeminska, I; Delgado, M; De Las Rivas, J; Ticona, F V; Fermiñán, E; Martín-Jiménez, P; Chillón, C; Risueño, A; Hernández, J M; García-Sanz, R; González, M; San Miguel, J F (2010).
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For the miRNA sequences were an exact match is found, obtain the genomic sequence including ~100bp of flanking sequence on either side, and run the RNA through RNA folding software such as the Vienna package.
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Novel miRNA sequences are identified based on the characteristic expression pattern that they display due to DICER processing: higher expression of the mature miRNA over the star strand and loop sequences.
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Each of the remaining sequences are aligned against a miRNA sequence database (such as miRBase) In order to account for imperfect DICER processing, a 6nt overhang on the 3’ end, and 3nt on the 5’ end are
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The shortlisted sequences are trimmed down to include only the possible precursor sequence and are then refolded to ensure that the precursor was not artificially stabilized by neighbouring sequences.
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Lowery, Aoife J; Miller, Nicola; Devaney, Amanda; McNeill, Roisin E; Davoren, Pamela A; Lemetre, Christophe; Benes, Vladimir; Schmidt, Sabine; Blake, Jonathon; Ball, Graham; Kerin, Michael J (2009).
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Blenkiron, Cherie; Goldstein, Leonard D; Thorne, Natalie P; Spiteri, Inmaculada; Chin, Suet-Feung; Dunning, Mark J; Barbosa-Morais, Nuno L; Teschendorff, Andrew E; Green, Andrew R; Ellis, Ian O;
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This step converts the small adaptor ligated RNAs into cDNA clones used in the sequencing reaction. There are many commercial kits available that will carry out this step using some form of
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Ratner, Elena S.; Tuck, David; Richter, Christine; Nallur, Sunitha; Patel, Rajeshvari M.; Schultz, Vince; Hui, Pei; Schwartz, Peter E.; Rutherford, Thomas J.; Weidhaas, Joanne B. (2010).
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technologies in order to find novel miRNAs and their expression profiles in a given sample. miRNA sequencing in and of itself is not a new idea, initial methods of sequencing utilized
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Starczynowski, D. T.; Morin, R.; McPherson, A.; Lam, J.; Chari, R.; Wegrzyn, J.; Kuchenbauer, F.; Hirst, M.; Tohyama, K.; Humphries, R. K.; Lam, W. L.; Marra, M.; Karsan, A. (2010).
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sequence information. Because of this, one can obtain sequences of novel miRNAs and miRNA isoforms (isoMirs), distinguish sequentially similar miRNAs, and identify point mutations.
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Observe for evidence of miRNA targeting in mRNA-seq or protein expression data: where the miRNA expression is high, the gene and protein expression of its target gene should be low.
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Zhang, Baohong; Zhang, Hua; Yang, Jian-Hua; Zheng, Yu-Sheng; Zhang, Peng; Chen, Xiao; Wu, Jun; Xu, Ling; Luo, Xue-Qun; Ke, Zhi-Yong; Zhou, Hui; Qu, Liang-Hu; Chen, Yue-Qin (2009).
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Weinberg, J. B.; Thompson, M. A.; Gill, J. I.; Liu, Q.; How, T.; Grubor, V.; Gao, Y.; Patel, A.; Wu, H.; Zhu, J.; Blobe, G. C.; Lipsky, P. E.; Chadburn, A.; Dave, S. S. (2010).
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The reads that do not align to the miRNA database are then loosely aligned to miRNA precursors to detect miRNAs that might carry mutations or those that have gone through
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Mattie, Michael D; Benz, Christopher C; Bowers, Jessica; Sensinger, Kelly; Wong, Linda; Scott, Gary K; Fedele, Vita; Ginzinger, David; Getts, Robert; Haqq, Chris (2006).
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Morin, R. D.; O'Connor, M. D.; Griffith, M.; Kuchenbauer, F.; Delaney, A.; Prabhu, A.-L.; Zhao, Y.; McDonald, H.; Zeng, T.; Hirst, M.; Eaves, C. J.; Marra, M. A. (2008).
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Determine the degree of conservation of miRNA:mRNA binding pairs across species. Typically, more highly binding pairs are less likely to be false positives of prediction.
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transcript and downregulated LIN-14 protein expression. miRNAs are now thought to be involved in the regulation of many developmental and biological processes, including
1827:"Ultra-high throughput sequencing-based small RNA discovery and discrete statistical biomarker analysis in a collection of cervical tumours and matched controls" 418:
The resulting folded sequences are considered novel miRNAs if the miRNA sequence falls within one arm of the hairpin, and are highly conserved between species.
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In a given sample all the RNA is extracted and isolated using an isothiocyanate/phenol/chloroform (GITC/phenol) method or a commercial product such as Trizol (
55:(miRNAs) are a family of small ribonucleic acids, 21-25 nucleotides in length, that modulate protein expression through transcript degradation, inhibition of 2403:
Hofacker, I. L.; Fontana, W.; Stadler, P. F.; Bonhoeffer, L. S.; Tacker, M.; Schuster, P. (1994). "Fast folding and comparison of RNA secondary structures".
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regulatory networks that may be driving a particular disorder. Several applications of miRNAs as biomarkers and predictors of disease are given below.
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Folded sequences that lie on one arm of the miRNA hairpin and have a minimum free energy of less than ~25kcal/mol are shortlisted as putative miRNA.
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Hafner, Markus; Landgraf, Pablo; Ludwig, Janos; Rice, Amanda; Ojo, Tolulope; Lin, Carolina; Holoch, Daniel; Lim, Cindy; Tuschl, Thomas (2008).
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Lu, C; Tej, SS; Luo, S; Haudenschild, CD; Meyers, BC; Green, PJ (Sep 2, 2005). "Elucidation of the small RNA component of the transcriptome".
3514:"Optimized high-throughput microRNA expression profiling provides novel biomarker assessment of clinical prostate and breast cancer biopsies" 3116:"Genome-Wide Analysis of Small RNA and Novel MicroRNA Discovery in Human Acute Lymphoblastic Leukemia Based on Extensive Sequencing Approach" 1685: 3920:"Deregulation of microRNA expression in the different genetic subtypes of multiple myeloma and correlation with gene expression profiling" 2105: 365:
After sequencing, the raw sequence reads are filtered based on quality. The adaptor sequences are also trimmed off the raw sequence reads.
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Berninger, Philipp; Gaidatzis, Dimos; van Nimwegen, Erik; Zavolan, Mihaela (2008). "Computational analysis of small RNA cloning data".
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The ligation step adds DNA adaptors to both ends of the small RNAs, which act as primer binding sites during reverse transcription and
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Sempere, L. F.; Christensen, M.; Silahtaroglu, A.; Bak, M.; Heath, C. V.; Schwartz, G.; Wells, W.; Kauppinen, S.; Cole, C. N. (2007).
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Ruby, J. Graham; Jan, Calvin; Player, Christopher; Axtell, Michael J.; Lee, William; Nusbaum, Chad; Ge, Hui; Bartel, David P. (2006).
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The actual RNA sequencing varies significantly depending on the platform used. Three common next-generation sequencing platforms are
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Rassenti, Laura; Alder, Hansjuerg; Volinia, Stefano; Liu, Chang-gong; Kipps, Thomas J.; Negrini, Massimo; Croce, Carlo M. (2005).
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Caramuta, Stefano; Egyházi, Suzanne; Rodolfo, Monica; Witten, Daniela; Hansson, Johan; Larsson, Catharina; Lui, Weng-Onn (2010).
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Determine miRNA:mRNA binding pairs, complementarity between the miRNA sequences at the 3’-UTR of the mRNA sequence is identified.
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Nic; Shepherd, Jill; Lee, Clarence; Ichikawa, Jeff; McKernan, Kevin; Bramlett, Kelli; Kuersten, Scott; Grimmond, Sean M (2011).
517:. Consequently, it is not unexpected that miRNAs are involved in various aspects of cancer through the regulation of onco- and 1550:
Johnston, Robert J.; Hobert, Oliver (2003). "A microRNA controlling left/right neuronal asymmetry in Caenorhabditis elegans".
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The read counts for each miRNA are then normalized to the total number of mapped miRNAs to report the abundance of each miRNA.
3187:"Deep sequencing of the small RNA transcriptome of normal and malignant human B cells identifies hundreds of novel microRNAs" 1367:"Posttranscriptional regulation of the heterochronic gene lin-14 by lin-4 mediates temporal pattern formation in C. elegans" 368:
The resulting reads are then formatted into a fasta file where the copy number and sequence is recorded for each unique tag.
1672:. Methods in Molecular Biology. Vol. 822. Next-Generation MicroRNA Expression Profiling Technology. pp. 19–31. 199:
SOLiD sequencing platform has also been used to examine the prognostic value of miRNAs in detecting human breast cancer.
128:). These discoveries necessitated development of techniques able to identify and characterize miRNAs, such as miRNA-seq. 3689:"Diagnostic Assay Based on hsa-miR-205 Expression Distinguishes Squamous From Nonsquamous Non-Small-Cell Lung Carcinoma" 1668:
Aldridge, Sarah; Hadfield, James (2012). "Introduction to miRNA Profiling Technologies and Cross-Platform Comparison".
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Ambros, Victor (1989). "A hierarchy of regulatory genes controls a larva-to-adult developmental switch in C. elegans".
3739:"Relation between microRNA expression and progression and prognosis of gastric cancer: a microRNA expression analysis" 3629:"MicroRNA signatures predict oestrogen receptor, progesterone receptor and HER2/neu receptor status in breast cancer" 1001:
Sandhu, S.; Garzon, R. (2011). "Potential Applications of MicroRNAs in Cancer Diagnosis, Prognosis, and Treatment".
2606:"Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets" 141: 137: 24: 2753:"Weak seed-pairing stability and high target-site abundance decrease the proficiency of lsy-6 and other microRNAs" 2144: 1491: 439: 281: 241: 79:
gene encoded a 22 nucleotide RNA with conserved complementary binding sites in the 3’-untranslated region of the
2557:"A pattern-based method for the identification of MicroRNA binding sites and their corresponding heteroduplexes" 2188:"Application of massively parallel sequencing to microRNA profiling and discovery in human embryonic stem cells" 272:
does not have to be necessarily carried out before the ligation and reverse transcription steps outlined below.
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or PARE. Validation of target cleavage in specific mRNAs is typically performed using a modified version of 5'
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Git, A.; Dvinge, H.; Salmon-Divon, M.; Osborne, M.; Kutter, C.; Hadfield, J.; Bertone, P.; Caldas, C. (2010).
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Lu, C; Meyers, BC; Green, PJ (October 2007). "Construction of small RNA cDNA libraries for deep sequencing".
4358: 112: 324: 183: 124: 71: 518: 372: 294: 260: 237: 191:. Another study comparing small RNA profiles of human cervical tumours and normal tissue, utilized the 56: 3055:
Buermans, Henk PJ; Ariyurek, Yavuz; van Ommen, Gertjan; den Dunnen, Johan T; 't Hoen, Peter AC (2010).
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Genome Analyzer to identify 64 novel human miRNA genes as well as 67 differentially expressed miRNAs.
1770:"Large-Scale Sequencing Reveals 21U-RNAs and Additional MicroRNAs and Endogenous siRNAs in C. elegans" 4040: 3127: 1726: 1618: 1559: 1430: 1318:"The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14" 480: 455:, miRanda, and PicTar are software designed for this purpose. A list of prediction software is given 328: 4029:"Distinctive microRNA signature of acute myeloid leukemia bearing cytoplasmic mutated nucleophosmin" 4196:"MicroRNA Expression Profiles Associated with Mutational Status and Survival in Malignant Melanoma" 229: 157: 3976:"MicroRNA expression profiling in relation to the genetic heterogeneity of acute myeloid leukemia" 3465:"Altered MicroRNA Expression Confined to Specific Epithelial Cell Subpopulations in Breast Cancer" 3244:"Genome-wide identification of human microRNAs located in leukemia-associated genomic alterations" 4277: 2988: 2635: 2586: 2428: 2087: 1807: 1750: 1650: 1591: 1532: 1472: 1298: 1247: 1191: 1134: 1080: 510: 320: 196: 192: 168:
miRNA-seq can be performed using a variety of sequencing platforms. The first analysis of small
4147:"A MicroRNA Signature Associated with Prognosis and Progression in Chronic Lymphocytic Leukemia" 1492:"The Drosophila MicroRNA Mir-14 Suppresses Cell Death and Is Required for Normal Fat Metabolism" 1214:
He, Lin; Hannon, Gregory J. (2004). "MicroRNAs: small RNAs with a big role in gene regulation".
2109: 4334: 4316: 4269: 4261: 4225: 4217: 4176: 4168: 4126: 4118: 4076: 4058: 4005: 3997: 3949: 3941: 3894: 3876: 3833: 3815: 3776: 3758: 3718: 3710: 3668: 3650: 3606: 3598: 3553: 3535: 3494: 3486: 3445: 3427: 3406:"MicroRNA expression profiling of human breast cancer identifies new markers of tumor subtype" 3379: 3361: 3317: 3309: 3273: 3265: 3224: 3206: 3163: 3145: 3096: 3078: 3037: 2980: 2935: 2927: 2892: 2841: 2823: 2782: 2733: 2684: 2627: 2578: 2537: 2519: 2477: 2469: 2420: 2385: 2367: 2328: 2310: 2268: 2260: 2225: 2207: 2163: 2079: 2071: 2033: 2015: 1967: 1925: 1907: 1866: 1848: 1799: 1791: 1742: 1699: 1691: 1681: 1642: 1634: 1583: 1575: 1524: 1516: 1464: 1456: 1415: 1396: 1388: 1347: 1339: 1290: 1282: 1239: 1231: 1183: 1175: 1126: 1118: 1072: 1064: 1018: 983: 965: 316: 145: 438:
of mapped reads between samples, one can use a host of statistical tests (like those used in
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screen to identify molecular elements controlling post-embryonic development of the nematode
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which identified 18 novel miRNA genes as well as a new class of nematode small RNAs termed
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using miRNA-seq methods examined approximately 1.4 million small RNAs from the model plant
118: 3008:"Endogenous siRNA and miRNA targets identified by sequencing of the Arabidopsis degradome" 2653:
Grimson, A; Farh, KK; Johnston, WK; Garrett-Engele, P; Lim, LP; Bartel, DP (Jul 6, 2007).
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3401: 2498:"MicroRNAs and their isomiRs function cooperatively to target common biological pathways" 4044: 3131: 1730: 1622: 1563: 1434: 343: 4329: 4296: 4071: 4028: 3889: 3856: 3828: 3795: 3771: 3738: 3663: 3628: 3548: 3513: 3440: 3405: 3374: 3341: 3219: 3186: 3158: 3115: 3091: 3056: 3032: 3007: 2887: 2860: 2836: 2801: 2777: 2752: 2728: 2703: 2679: 2654: 2532: 2497: 2380: 2347: 2323: 2290: 2220: 2187: 2028: 1995: 1920: 1885: 1861: 1826: 978: 945: 509:
Micro RNAs are important regulators of almost all cellular processes such as survival,
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reverse transcribed from endogenous small RNAs of 21–25 bp size selected by column and
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Obtain reads that did not align to known miRNA sequences, and map them to the genome.
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Witten, Daniela; Tibshirani, Robert; Gu, Sam; Fire, Andrew; Lui, Weng-Onn (2010).
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Garcia, DM; Baek, D; Shin, C; Bell, GW; Grimson, A; Bartel, DP (Sep 11, 2011).
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integrated to mRNA-seq data to observe for miRNA:mRNA functional pairs. RNA22,
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Agarwal, Vikram; Bell, George W.; Nam, Jin-Wu; Bartel, David P. (2015-08-12).
1101:; Han, Jinju; Siomi, Mikiko C. (2009). "Biogenesis of small RNAs in animals". 452: 256: 233: 4320: 4265: 4221: 4172: 4122: 4062: 4001: 3945: 3880: 3857:"Accurate Molecular Classification of Renal Tumors Using MicroRNA Expression" 3819: 3762: 3714: 3705: 3688: 3654: 3602: 3539: 3490: 3431: 3365: 3313: 3269: 3210: 3149: 3082: 3073: 2931: 2859:
Agarwal, V; Subtelny, AO; Thiru, P; Ulitsky, I; Bartel, DP (4 October 2018).
2827: 2655:"MicroRNA targeting specificity in mammals: determinants beyond seed pairing" 2523: 2473: 2424: 2371: 2314: 2264: 2211: 2075: 2019: 1911: 1852: 1795: 1695: 1638: 1579: 1520: 1460: 1392: 1343: 1286: 1235: 1179: 1122: 1068: 969: 944:
Farazi, Thalia A; Spitzer, Jessica I; Morozov, Pavel; Tuschl, Thomas (2011).
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This is not a comprehensive list of miRNAs involved with these malignancies.
4053: 2130: 1738: 1630: 1442: 4338: 4273: 4229: 4180: 4130: 4080: 4009: 3953: 3898: 3837: 3780: 3722: 3672: 3610: 3557: 3530: 3498: 3449: 3383: 3321: 3277: 3228: 3167: 3100: 3041: 2984: 2939: 2896: 2845: 2786: 2737: 2688: 2631: 2582: 2541: 2481: 2389: 2332: 2272: 2229: 2083: 2037: 1971: 1929: 1884:
Wu, Qian; Lu, Zuhong; Li, Hailing; Lu, Jiafeng; Guo, Li; Ge, Qinyu (2011).
1870: 1843: 1803: 1746: 1703: 1646: 1587: 1528: 1468: 1243: 1187: 1130: 1076: 1022: 987: 4257: 2719: 2363: 1902: 1400: 1351: 1294: 1059: 1042: 4163: 4146: 4113: 4096: 3936: 3919: 2306: 52: 32: 4312: 4212: 4195: 4095:
Caligiuri, Michael A.; Larson, Richard A.; Bloomfield, Clara D. (2008).
2818: 1571: 3305: 2416: 2203: 1451: 371:
Sequences that may represent E. Coli contamination are identified by a
188: 149: 20: 4097:"MicroRNA Expression in Cytogenetically Normal Acute Myeloid Leukemia" 2768: 2148: 1490:
Xu, Peizhang; Vernooy, Stephanie Y.; Guo, Ming; Hay, Bruce A. (2003).
961: 550:
miR-26a/b, miR-30 family, miR-29b, miR-155, miR-342, miR-206, miR-191
1886:"Next-Generation Sequencing of MicroRNAs for Breast Cancer Detection" 358: 36: 3645: 3340:
Chan, Elcie; Prado, Daniel Estévez; Weidhaas, Joanne Barnes (2011).
2976: 2067: 2054:
Shendure, Jay; Ji, Hanlee (2008). "Next-generation DNA sequencing".
1227: 1114: 2348:"miRBase: integrating microRNA annotation and deep-sequencing data" 635:
miR-19a/b, miR-30e-5p, miR-101, miR-452, miR-382, miR-15a, miR-29c
136:
MicroRNA sequencing (miRNA-seq) was developed to take advantage of
342: 206: 3796:"MicroRNA signatures differentiate uterine cancer tumor subtypes" 2952:
Krek, A. Identification of microRNA targets. DAI-B 70/07, (2010).
2910:
Maziere, P; Enright, A (2007). "Prediction of microRNA targets".
375:
search against an E. Coli database and are removed from analysis.
178:
Lynx Therapeutics' Massively Parallel Signature Sequencing (MPSS)
59:, or sequestering transcripts. The first miRNA to be discovered, 148:
methods. Sequencing preparation involved creating libraries by
85: 2802:"Predicting effective microRNA target sites in mammalian mRNAs" 3577:"MicroRNA Gene Expression Deregulation in Human Breast Cancer" 2702:
Friedman, RC; Farh, KK; Burge, CB; Bartel, DP (January 2009).
1316:
Lee, Rosalind C.; Feinbaum, Rhonda L.; Ambros, Victor (1993).
169: 153: 3969: 3967: 3965: 3963: 3912: 3910: 3908: 1416:"MicroRNAs Modulate Hematopoietic Lineage Differentiation" 3395: 3393: 2704:"Most mammalian mRNAs are conserved targets of microRNAs" 716:
miR-382, miR-134, miR-376a, miR-127, miR-299-5p, miR-323
357:
absolute abundance, to discover their variants (known as
2289:
Creighton, C. J.; Reid, J. G.; Gunaratne, P. H. (2009).
2181: 2179: 1670:
Next-Generation MicroRNA Expression Profiling Technology
1154:"MicroRNAsGenomics, Biogenesis, Mechanism, and Function" 740:
miR-10a/b, let-7, miR-29, miR-204, miR-128a, miR-196a/b
319:
platform, polymerase-based sequence-by-synthesis on the
267:
Size Fractionation of small RNAs by Gel Electrophoresis
2861:"Predicting microRNA targeting efficacy in Drosophila" 2291:"Expression profiling of microRNAs by deep sequencing" 4021: 4019: 3006:
Addo-Quaye C, Eshoo TW, Bartel DP, Axtell MJ (2008).
1989: 1987: 1985: 1983: 1981: 2284: 2282: 526:
Table 1: Cancer subtypes distinguished by microRNAs
3849: 3847: 3622: 3620: 939: 937: 1043:"MicroRNA profiling: separating signal from noise" 560:let-7c, miR-29b, miR-26a, miR-30 family, miR-520g 2049: 2047: 3335: 3333: 3331: 3179: 3177: 1365:Wightman, Bruce; Ha, Ilho; Ruvkun, Gary (1993). 838:Table 2: Platform Comparison of miRNA Profiling 818:Comparison With Other Methods of miRNA Profiling 574:miR-520d, miR-181c, miR-302c, miR-376b, miR-30e 4033:Proceedings of the National Academy of Sciences 2604:Lewis, BP; Burge CB; Bartel DP (Jan 14, 2005). 1945: 1943: 1941: 1939: 1209: 1207: 1205: 352:miRNA Alignment & Abundance Quantification 8: 3569: 3567: 771:miR-15a, miR-195, miR-221, miR-155, miR-23b 1036: 1034: 1032: 289:Reverse Transcription and PCR Amplification 2346:Kozomara, A.; Griffiths-Jones, S. (2010). 898:Infrastructure and technical requirements 474:Target Validation for Cleaved mRNA Targets 4328: 4211: 4162: 4112: 4070: 4052: 3991: 3935: 3888: 3827: 3770: 3704: 3662: 3644: 3592: 3547: 3529: 3480: 3439: 3421: 3373: 3259: 3218: 3157: 3139: 3090: 3072: 3031: 2886: 2876: 2835: 2817: 2776: 2757:Nature Structural & Molecular Biology 2727: 2678: 2621: 2572: 2531: 2513: 2463: 2379: 2322: 2219: 2027: 1919: 1901: 1860: 1842: 1785: 1510: 1450: 1382: 1333: 1169: 1058: 977: 3404:; Caldas, Carlos; Miska, Eric A (2007). 1890:Journal of Biomedicine and Biotechnology 836: 524: 423:Star Strand Expression Method (miRdeep) 40:requirements, run length, and potential 933: 442:) to determine differential expression 2168:: CS1 maint: archived copy as title ( 2161: 831:Platform Comparison of miRNA Profiling 27:or massively parallel high-throughput 1103:Nature Reviews Molecular Cell Biology 618:miR-29b/c, miR-30 family, miR-135a/b 7: 4200:Journal of Investigative Dermatology 3861:The Journal of Molecular Diagnostics 794: 777: 760: 705: 668: 641: 624: 607: 580: 541: 285:products with a 5’ hydroxyl group. 652:miR-424, miR-203, miR-31, miR-126 632:Endometrioid vs uterine papillary 601:miR-17-5p, miR-22, miR-24, miR-31 14: 893:Five or more orders of magnitude 181:technologies, such as a study in 1015:10.1053/j.seminoncol.2011.08.007 485:Rapid Amplification of cDNA Ends 433:Differential Expression Analysis 4151:New England Journal of Medicine 4101:New England Journal of Medicine 17:MicroRNA sequencing (miRNA-seq) 802:Diffuse Large B Cell Lymphoma 768:ZAP-70 levels and IgVH status 588:Squamous vs non-squamous cell 496:Identification of Novel miRNAs 1: 3755:10.1016/S1470-2045(09)70343-2 3594:10.1158/0008-5472.CAN-05-1783 3482:10.1158/0008-5472.CAN-07-5019 2465:10.1093/bioinformatics/btr430 1512:10.1016/S0960-9822(03)00250-1 1171:10.1016/S0092-8674(04)00045-5 598:Small cell vs non-small cell 487:with a gene-specific primer. 3993:10.1182/blood-2008-01-133355 3693:Journal of Clinical Oncology 3358:10.1016/j.molmed.2011.01.008 3346:Trends in Molecular Medicine 3261:10.1182/blood-2010-03-277012 3203:10.1182/blood-2010-05-285403 3141:10.1371/journal.pone.0006849 2924:10.1016/j.drudis.2007.04.002 2671:10.1016/j.molcel.2007.06.017 1384:10.1016/0092-8674(93)90530-4 1335:10.1016/0092-8674(93)90529-Y 1279:10.1016/0092-8674(89)90171-2 762:Chronic lymphocytic leukemia 226:Phenol–chloroform extraction 116:) and neuronal development ( 3812:10.1016/j.ygyno.2010.05.010 2295:Briefings in Bioinformatics 2257:10.1016/j.ymeth.2007.10.002 2012:10.1016/j.ymeth.2007.09.009 1964:10.1016/j.ymeth.2007.05.002 1678:10.1007/978-1-61779-427-8_2 788:miR-193a, miR-338, miR-565 699:miR-125a, miR-650, miR-184 4375: 3873:10.2353/jmoldx.2010.090187 3292:of lung cancer patients". 2623:10.1016/j.cell.2004.12.035 2574:10.1016/j.cell.2006.07.031 2515:10.1186/gb-2011-12-12-r126 1787:10.1016/j.cell.2006.10.040 689:miR-203, miR-155, miR-375 659:Oncocytoma vs chromophobe 304: 219: 142:high-throughput sequencing 138:next-generation sequencing 25:next-generation sequencing 3423:10.1186/gb-2007-8-10-r214 3024:10.1016/j.cub.2008.04.042 2878:10.1186/s13059-018-1504-3 2446:Yang, X.; Li, L. (2011). 911: 897: 890:Four orders of magnitude 883: 869: 855: 850: 847: 844: 842: 459:. The general steps are: 440:gene expression profiling 242:Polymerase chain reaction 211:miRNA Library Preparation 65:, was found in a genetic 3706:10.1200/JCO.2008.19.4134 3074:10.1186/1471-2164-11-716 950:The Journal of Pathology 946:"miRNAs in human cancer" 887:Six orders of magnitude 723:with t(8;21) or inv(16) 649:Clear cell vs papillary 4054:10.1073/pnas.0800135105 2358:(Database): D152–D157. 2131:"Illumina DesignStudio" 1739:10.1126/science.1114112 1631:10.1126/science.1065329 1443:10.1126/science.1091903 1216:Nature Reviews Genetics 884:Dynamic range detected 347:miRNA-seq Data Analysis 113:Drosophila melanogaster 3633:Breast Cancer Research 3531:10.1186/1476-4598-5-24 2405:Monatshefte für Chemie 2352:Nucleic Acids Research 1844:10.1186/1741-7007-8-58 912:Cost per sample (USD) 707:Acute myeloid leukemia 615:Diffuse vs intestinal 348: 325:sequencing by ligation 212: 140:or massively parallel 125:Caenorhabditis elegans 72:Caenorhabditis elegans 4258:10.1038/nmeth0709-474 2720:10.1101/gr.082701.108 1060:10.1038/nmeth0910-687 1041:Baker, Monya (2010). 662:miR-200c, miR-139-5p 519:tumor suppressor gene 394:Novel miRNA Discovery 346: 295:reverse transcriptase 261:Caliper Life Sciences 238:Reverse transcriptase 216:Small RNA Preparation 210: 104:), lipid metabolism ( 4164:10.1056/NEJMoa050995 4114:10.1056/NEJMoa074256 3937:10.1038/leu.2009.274 3800:Gynecologic Oncology 3294:Molecular BioSystems 2912:Drug Discovery Today 2056:Nature Biotechnology 1414:Chen, C.-Z. (2004). 481:Degradome sequencing 329:ABI Solid Sequencing 174:Arabidopsis thaliana 4313:10.1261/rna.1947110 4213:10.1038/jid.2010.63 4045:2008PNAS..105.3945G 3743:The Lancet Oncology 3475:(24): 11612–11620. 3132:2009PLoSO...4.6849Z 2819:10.7554/eLife.05005 2364:10.1093/nar/gkq1027 1903:10.1155/2011/597145 1731:2005Sci...309.1567L 1623:2001Sci...294..862L 1572:10.1038/nature02255 1564:2003Natur.426..845J 1435:2004Sci...303...83C 870:Total RNA required 839: 527: 406:RNA Folding Method 251:Total RNA Isolation 230:Gel Electrophoresis 158:gel electrophoresis 3306:10.1039/c1mb05353a 2918:(11–12): 452–458. 2417:10.1007/BF00818163 2307:10.1093/bib/bbp019 2204:10.1101/gr.7179508 1152:Bartel, D (2004). 879:500-5,000 ng 876:100-1,000 ng 837: 726:let-7b/c, miR-127 525: 505:Disease biomarkers 349: 321:Illumina (company) 213: 197:Applied Biosystems 193:Illumina (company) 4157:(17): 1793–1801. 4107:(18): 1919–1928. 4039:(10): 3945–3950. 3986:(10): 5078–5085. 3699:(12): 2030–2037. 3587:(16): 7065–7070. 3197:(23): e118–e127. 2769:10.1038/nsmb.2115 2062:(10): 1135–1145. 1687:978-1-61779-426-1 1617:(5543): 862–864. 1558:(6968): 845–849. 962:10.1002/path.2806 925: 924: 812: 811: 446:Target Prediction 317:454 Life Sciences 146:Sanger sequencing 4366: 4343: 4342: 4332: 4292: 4286: 4285: 4240: 4234: 4233: 4215: 4206:(8): 2062–2070. 4191: 4185: 4184: 4166: 4141: 4135: 4134: 4116: 4091: 4085: 4084: 4074: 4056: 4023: 4014: 4013: 3995: 3971: 3958: 3957: 3939: 3914: 3903: 3902: 3892: 3851: 3842: 3841: 3831: 3791: 3785: 3784: 3774: 3733: 3727: 3726: 3708: 3683: 3677: 3676: 3666: 3648: 3624: 3615: 3614: 3596: 3571: 3562: 3561: 3551: 3533: 3518:Molecular Cancer 3509: 3503: 3502: 3484: 3460: 3454: 3453: 3443: 3425: 3397: 3388: 3387: 3377: 3337: 3326: 3325: 3288: 3282: 3281: 3263: 3239: 3233: 3232: 3222: 3181: 3172: 3171: 3161: 3143: 3111: 3105: 3104: 3094: 3076: 3052: 3046: 3045: 3035: 3003: 2997: 2996: 2959: 2953: 2950: 2944: 2943: 2907: 2901: 2900: 2890: 2880: 2856: 2850: 2849: 2839: 2821: 2797: 2791: 2790: 2780: 2748: 2742: 2741: 2731: 2699: 2693: 2692: 2682: 2650: 2644: 2643: 2625: 2601: 2595: 2594: 2576: 2552: 2546: 2545: 2535: 2517: 2492: 2486: 2485: 2467: 2443: 2437: 2436: 2400: 2394: 2393: 2383: 2343: 2337: 2336: 2326: 2286: 2277: 2276: 2240: 2234: 2233: 2223: 2183: 2174: 2173: 2167: 2159: 2157: 2156: 2147:. Archived from 2141: 2135: 2134: 2127: 2121: 2120: 2118: 2117: 2108:. Archived from 2102: 2096: 2095: 2051: 2042: 2041: 2031: 1991: 1976: 1975: 1947: 1934: 1933: 1923: 1905: 1881: 1875: 1874: 1864: 1846: 1822: 1816: 1815: 1789: 1780:(6): 1193–1207. 1765: 1759: 1758: 1725:(5740): 1567–9. 1714: 1708: 1707: 1665: 1659: 1658: 1606: 1600: 1599: 1547: 1541: 1540: 1514: 1496: 1487: 1481: 1480: 1454: 1420: 1411: 1405: 1404: 1386: 1362: 1356: 1355: 1337: 1313: 1307: 1306: 1262: 1256: 1255: 1211: 1200: 1199: 1173: 1149: 1143: 1142: 1095: 1089: 1088: 1062: 1038: 1027: 1026: 998: 992: 991: 981: 941: 856:Throughput time 840: 785:with BRAF V600E 679:miR-1, miR-133a 528: 23:, is the use of 4374: 4373: 4369: 4368: 4367: 4365: 4364: 4363: 4349: 4348: 4347: 4346: 4307:(5): 991–1006. 4294: 4293: 4289: 4242: 4241: 4237: 4193: 4192: 4188: 4143: 4142: 4138: 4093: 4092: 4088: 4025: 4024: 4017: 3973: 3972: 3961: 3916: 3915: 3906: 3853: 3852: 3845: 3793: 3792: 3788: 3735: 3734: 3730: 3685: 3684: 3680: 3646:10.1186/bcr2257 3626: 3625: 3618: 3581:Cancer Research 3573: 3572: 3565: 3511: 3510: 3506: 3469:Cancer Research 3462: 3461: 3457: 3399: 3398: 3391: 3339: 3338: 3329: 3300:(12): 3187–99. 3290: 3289: 3285: 3241: 3240: 3236: 3183: 3182: 3175: 3113: 3112: 3108: 3054: 3053: 3049: 3018:(10): 758–762. 3005: 3004: 3000: 2977:10.1038/nbt1417 2965:Nat. Biotechnol 2961: 2960: 2956: 2951: 2947: 2909: 2908: 2904: 2858: 2857: 2853: 2799: 2798: 2794: 2763:(10): 1139–46. 2750: 2749: 2745: 2708:Genome Research 2701: 2700: 2696: 2652: 2651: 2647: 2603: 2602: 2598: 2554: 2553: 2549: 2494: 2493: 2489: 2445: 2444: 2440: 2402: 2401: 2397: 2345: 2344: 2340: 2288: 2287: 2280: 2242: 2241: 2237: 2192:Genome Research 2185: 2184: 2177: 2160: 2154: 2152: 2145:"Archived copy" 2143: 2142: 2138: 2129: 2128: 2124: 2115: 2113: 2104: 2103: 2099: 2068:10.1038/nbt1486 2053: 2052: 2045: 1993: 1992: 1979: 1949: 1948: 1937: 1883: 1882: 1878: 1824: 1823: 1819: 1767: 1766: 1762: 1716: 1715: 1711: 1688: 1667: 1666: 1662: 1608: 1607: 1603: 1549: 1548: 1544: 1499:Current Biology 1494: 1489: 1488: 1484: 1429:(5654): 83–86. 1418: 1413: 1412: 1408: 1364: 1363: 1359: 1315: 1314: 1310: 1264: 1263: 1259: 1228:10.1038/nrg1379 1213: 1212: 1203: 1151: 1150: 1146: 1115:10.1038/nrm2632 1097: 1096: 1092: 1040: 1039: 1030: 1000: 999: 995: 943: 942: 935: 930: 833: 820: 515:differentiation 507: 498: 493: 476: 448: 435: 396: 354: 337: 309: 303: 244: 218: 205: 134: 50: 12: 11: 5: 4372: 4370: 4362: 4361: 4359:DNA sequencing 4351: 4350: 4345: 4344: 4287: 4252:(7): 474–476. 4246:Nature Methods 4235: 4186: 4136: 4086: 4015: 3959: 3930:(3): 629–637. 3904: 3867:(5): 687–696. 3843: 3806:(3): 251–257. 3786: 3749:(2): 136–146. 3728: 3678: 3616: 3563: 3504: 3455: 3410:Genome Biology 3389: 3352:(5): 235–243. 3327: 3283: 3254:(2): 595–607. 3234: 3173: 3106: 3047: 2998: 2971:(8): 941–946. 2954: 2945: 2902: 2865:Genome Biology 2851: 2792: 2743: 2694: 2659:Molecular Cell 2645: 2596: 2567:(6): 1203–17. 2547: 2502:Genome Biology 2487: 2458:(18): 2614–5. 2452:Bioinformatics 2438: 2411:(2): 167–188. 2395: 2338: 2301:(5): 490–497. 2278: 2235: 2198:(4): 610–621. 2175: 2136: 2122: 2097: 2043: 1977: 1935: 1876: 1817: 1760: 1709: 1686: 1660: 1601: 1542: 1505:(9): 790–795. 1482: 1406: 1377:(5): 855–862. 1357: 1328:(5): 843–854. 1308: 1257: 1222:(7): 522–531. 1201: 1164:(2): 281–297. 1144: 1109:(2): 126–139. 1090: 1053:(9): 687–692. 1047:Nature Methods 1028: 1009:(6): 781–787. 993: 956:(2): 102–115. 932: 931: 929: 926: 923: 922: 919: 916: 913: 909: 908: 905: 902: 899: 895: 894: 891: 888: 885: 881: 880: 877: 874: 871: 867: 866: 863: 860: 857: 853: 852: 849: 846: 843: 832: 829: 819: 816: 810: 809: 807: 803: 799: 798: 792: 791: 789: 786: 782: 781: 775: 774: 772: 769: 765: 764: 758: 757: 755: 752: 744: 743: 741: 738: 730: 729: 727: 724: 720: 719: 717: 714: 713:with t(15;17) 710: 709: 703: 702: 700: 697: 696:with t(11;14) 693: 692: 690: 687: 683: 682: 680: 677: 676:with t(14;16) 673: 672: 666: 665: 663: 660: 656: 655: 653: 650: 646: 645: 639: 638: 636: 633: 629: 628: 622: 621: 619: 616: 612: 611: 605: 604: 602: 599: 595: 594: 592: 589: 585: 584: 578: 577: 575: 572: 564: 563: 561: 558: 554: 553: 551: 548: 544: 543: 539: 538: 535: 532: 506: 503: 497: 494: 492: 489: 475: 472: 471: 470: 467: 464: 447: 444: 434: 431: 430: 429: 428: 427: 421: 420: 419: 416: 413: 410: 404: 395: 392: 391: 390: 387: 380: 376: 369: 366: 353: 350: 336: 333: 313:Pyrosequencing 307:DNA sequencing 302: 299: 222:RNA extraction 217: 214: 204: 201: 162:DNA microarray 133: 130: 90:haematopoiesis 49: 46: 29:DNA sequencing 13: 10: 9: 6: 4: 3: 2: 4371: 4360: 4357: 4356: 4354: 4340: 4336: 4331: 4326: 4322: 4318: 4314: 4310: 4306: 4302: 4298: 4291: 4288: 4283: 4279: 4275: 4271: 4267: 4263: 4259: 4255: 4251: 4247: 4239: 4236: 4231: 4227: 4223: 4219: 4214: 4209: 4205: 4201: 4197: 4190: 4187: 4182: 4178: 4174: 4170: 4165: 4160: 4156: 4152: 4148: 4140: 4137: 4132: 4128: 4124: 4120: 4115: 4110: 4106: 4102: 4098: 4090: 4087: 4082: 4078: 4073: 4068: 4064: 4060: 4055: 4050: 4046: 4042: 4038: 4034: 4030: 4022: 4020: 4016: 4011: 4007: 4003: 3999: 3994: 3989: 3985: 3981: 3977: 3970: 3968: 3966: 3964: 3960: 3955: 3951: 3947: 3943: 3938: 3933: 3929: 3925: 3921: 3913: 3911: 3909: 3905: 3900: 3896: 3891: 3886: 3882: 3878: 3874: 3870: 3866: 3862: 3858: 3850: 3848: 3844: 3839: 3835: 3830: 3825: 3821: 3817: 3813: 3809: 3805: 3801: 3797: 3790: 3787: 3782: 3778: 3773: 3768: 3764: 3760: 3756: 3752: 3748: 3744: 3740: 3732: 3729: 3724: 3720: 3716: 3712: 3707: 3702: 3698: 3694: 3690: 3682: 3679: 3674: 3670: 3665: 3660: 3656: 3652: 3647: 3642: 3638: 3634: 3630: 3623: 3621: 3617: 3612: 3608: 3604: 3600: 3595: 3590: 3586: 3582: 3578: 3570: 3568: 3564: 3559: 3555: 3550: 3545: 3541: 3537: 3532: 3527: 3523: 3519: 3515: 3508: 3505: 3500: 3496: 3492: 3488: 3483: 3478: 3474: 3470: 3466: 3459: 3456: 3451: 3447: 3442: 3437: 3433: 3429: 3424: 3419: 3415: 3411: 3407: 3403: 3402:Tavaré, Simon 3396: 3394: 3390: 3385: 3381: 3376: 3371: 3367: 3363: 3359: 3355: 3351: 3347: 3343: 3336: 3334: 3332: 3328: 3323: 3319: 3315: 3311: 3307: 3303: 3299: 3295: 3287: 3284: 3279: 3275: 3271: 3267: 3262: 3257: 3253: 3249: 3245: 3238: 3235: 3230: 3226: 3221: 3216: 3212: 3208: 3204: 3200: 3196: 3192: 3188: 3180: 3178: 3174: 3169: 3165: 3160: 3155: 3151: 3147: 3142: 3137: 3133: 3129: 3125: 3121: 3117: 3110: 3107: 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