We thank the entire Sigma Life Science Mission Target ID Library development team, especially Kevin Gutshall for finding the technology and negotiating license terms, and Heather Holemon for her support and participation in fruitful troubleshooting discussions. We also thank Dr. Joop G&auml;ken of King&#x2019;s College London for freely sharing unpublished results and suggestions, and Qazi Hamid of RxBiosciences for preparing a great batch of cDNA and his persistence in getting it cloned. We would like to acknowledge Nan Lin and Scott Bahr from SAFC for performing cDNA arrays and data analysis.
MISSION is a registered trademark of Sigma-Aldrich Biotechnology L.P.

<ol>
	<li>Huang, Q., Gumireddy, K., Schrier, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+microRNAs+miR-373+and+miR-520c+promote+tumour+invasion+and+metastasis.">The microRNAs miR-373 and miR-520c promote tumour invasion and metastasis.</a> Nat. Cell Biol. 10 (2), 202-210 (2008).</li><li>Lichner, Z., Pall, E., Kerekes, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+miR-290-295+cluster+promotes+pluripotency+maintenance+by+regulating+cell+cycle+phase+distribution+in+mouse+embryonic+stem+cells.">The miR-290-295 cluster promotes pluripotency maintenance by regulating cell cycle phase distribution in mouse embryonic stem cells.</a> Differentiation. 81 (1), 11-24 (2011).</li><li>Subramanyam, D., Lamouille, S., Judson, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+targets+of+miR-302+and+miR-372+promote+reprogramming+of+human+fibroblasts+to+induced+pluripotent+stem+cells.">Multiple targets of miR-302 and miR-372 promote reprogramming of human fibroblasts to induced pluripotent stem cells.</a> Nat. Biotechnol. 29 (5), 443-448 (2001).</li><li>Lim, L. P., Lau, N. C., Garrett-Engele, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microarray+analysis+shows+that+some+microRNAs+downregulate+large+numbers+of+target+mRNAs.">Microarray analysis shows that some microRNAs downregulate large numbers of target mRNAs.</a> Nature. 433 (7027), 769-773 (2005).</li><li>Huntzinger, E., Izaurralde, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+silencing+by+microRNAs:+contributions+of+translational+repression+and+mRNA+decay.">Gene silencing by microRNAs: contributions of translational repression and mRNA decay.</a> Nat. Rev. Genet. 12 (2), 99-110 (2011).</li><li>Thomas, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Desperately+seeking+microRNA+targets.">Desperately seeking microRNA targets.</a> Nat. Struc. Mol. Biol. 17 (10), 1169-1174 (2010).</li><li>Baek, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+impact+of+microRNAs+on+protein+output.">The impact of microRNAs on protein output.</a> Nature. 455 (7209), 71-71 (2008).</li><li>Guo, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mammalian+microRNAs+predominantly+act+to+decrease+target+mRNA+levels.">Mammalian microRNAs predominantly act to decrease target mRNA levels.</a> Nature. 466 (3708), 835-840 (2010).</li><li>Ma, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumour+invasion+and+metastasis+initiated+my+microRNA-10b+in+breast+cancer.">Tumour invasion and metastasis initiated my microRNA-10b in breast cancer.</a> Nature. 449 (7163), 682-688 (2007).</li><li>Visone, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNAs+miR-221+and+miR-222,+both+overexpressed+in+human+thyroid+papillary+carcinomas,+regulate+p27Kip1+protein+levels+and+cell+cycle.">MicroRNAs miR-221 and miR-222, both overexpressed in human thyroid papillary carcinomas, regulate p27Kip1 protein levels and cell cycle.</a> Endocrine-Related Cancer. 14 (3), 791-798 (2007).</li><li>Lu, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA-21+promotes+cell+transformation+by+targeting+the+programmed+cell+death+4+gene.">MicroRNA-21 promotes cell transformation by targeting the programmed cell death 4 gene.</a> Oncogene. 27 (31), 4373-4379 (2008).</li><li>Guo, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+noncoding+RNA,+miR-126,+suppresses+the+growth+of+neoplastic+cells+by+targeting+phosphatidylinositol+3-kinase+signaling+and+is+frequently+lost+in+colon+cancers.">The noncoding RNA, miR-126, suppresses the growth of neoplastic cells by targeting phosphatidylinositol 3-kinase signaling and is frequently lost in colon cancers.</a> Genes Chrom. Cancer. 47 (11), 939-946 (2008).</li><li>Yamakuchi, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=miR-34a+repression+of+SIRT1+regulates+apoptosis.">miR-34a repression of SIRT1 regulates apoptosis.</a> Proc. Natl. Acad. Sci. U.S.A. 105 (36), 13421-13426 (2008).</li><li>Wickramasinghe, N. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estradiol+downregulates+miR-21+expression+and+increases+miR-21+target+gene+expression+in+MCF-7+breast+cancer+cells.">Estradiol downregulates miR-21 expression and increases miR-21 target gene expression in MCF-7 breast cancer cells.</a> Nucleic Acid Res. 37 (8), 2584-2595 .</li><li>Ciaudo, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+dynamic+and+sex-specific+expression+of+microRNAs+during+early+ES+cell+differentiation.">Highly dynamic and sex-specific expression of microRNAs during early ES cell differentiation.</a> PLoS Genetics. 5, e1000620 (2009).</li><li>Vallejo, D. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+Notch+signaling+by+the+conserved+miR-8/200+microRNA+family+in+development+and+cancer+cells.">Targeting Notch signaling by the conserved miR-8/200 microRNA family in development and cancer cells.</a> EMBO J. 30 (4), 756-769 (2011).</li><li>Jafarifar, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repression+of+VEGFA+by+CA-rich+element-binding+microRNAs+is+modulated+by+hnRNAP.">Repression of VEGFA by CA-rich element-binding microRNAs is modulated by hnRNAP.</a> L. EMBO J. 30 (7), 1324-1334 (2011).</li><li>Clancy, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=mRNA+isoform+diversity+can+obscure+detection+of+miRNA-mediated+control+of+translation.">mRNA isoform diversity can obscure detection of miRNA-mediated control of translation.</a> RNA. 17 (6), 1025-1033 (2011).</li></ol>

Authors declare financial relationships with commercial entities that have an interest in the submitted work.

microRNAs are 20-24 nucleotide RNAs that regulate gene expression post-transcriptionally by inhibiting mRNA translation, and frequently, destabilizing the targeted mRNA (reviewed in [6]). A single miRNA may regulate several hundred mRNAs to control a cell&#x2019;s response to developmental and environmental signals. Identifying and validating target mRNAs is essential in determining a miRNA&#x2019;s role and function in these pathways. However, target identification is not straightforward because, in animals, miRNAs and their target sites are not fully complementary. The &#x201C;seed&#x201D; region, bases 2 through 7 from the 5&#x2032;-end of the miRNA, is usually complementary to its targets. However, there are many exceptions to the seed rule, and downstream base-pairing can compensate for an imperfect seed match. A number of computer algorithms have been developed to predict miRNA targets based on seed matching and downstream compensation, target structure and position, sequence conservation, and various other parameters that have been observed for experimentally validated targets (reviewed in [7]). While in silico predictions are convenient and do identify many valid miRNA targets, most predicted genes fail experimental validation tests, and many actual targets are not predicted. Furthermore, since computer algorithms are based on previously determined target features, they do not allow discovery of targets that deviate from what is already known.

A number of experimental systems have been used successfully to identify or discover functional miRNA targets in living cells. These global screening methods include microarrays and RNA sequencing, RNA co-immunoprecipitation (RIP), and Stable Isotope Labeling with Amino acids in Cell culture (SILAC), a proteomic method (reviewed in [7]). Each method has advantages and disadvantages. Since miRNA targeting often destabilizes an mRNA and leads to its degradation, loss or gain in mRNA level after introducing a miRNA mimic or inhibitor, respectively, can identify miRNA targets. This loss or gain of mRNA is readily detected by microarray or deep sequencing. While mRNA detection methods are simpler and more sensitive than protein detection methods, mRNA detection will miss any miRNA targets that are not degraded. Recent reports from the Bartell lab comparing miRNA target results from RNA detection with those from SILAC [8] or ribosome profiling [9] indicate that mammalian miRNAs predominantly act by reducing target mRNA levels. However, many other labs working with individual miRNA targets have detected change in protein but no change in mRNA level. Reports of what appear to be translational regulation with no detectable mRNA loss include: miR-10b on HOXD10 [10], miR221/222 on p27kip1 [11], miR-21 on Pdcd4 [12], miR-126 on p85&#x03B2; [13], miR-34 on SIRT1 [14], miR-21 on PTEN [15], miR-302d on Arid4b [16], miR-200c on JAG1 [17], and miR-299, 297, 567, and 609 on VEGFA [18]. Furthermore, Clancy et al [19] recently reported that translational regulation by let-7 was only detected when individual mRNA isoforms that contain the let-7 target site were detected selectively. The latter suggests that, at least in some cases, translational regulation may be overlooked in a composite profile &#x2013; that is, when all mRNA isoforms are detected as one mRNA &#x2013; as obtained with many microarray and sequence analyses. Co-immunoprecipitation of miRNA-mRNA complexes via argonaute (usually ago2) or another associated protein (RNA immunoprecipitation, or RIP) will isolate miRNA targets regardless of regulation mechanism. In addition, RIP detects endogenous, and presumably biologically relevant, interactions. However, it is not known whether all miRNA-mRNA interactions are functional, and RIP would miss any mRNA targets that associate only transiently or are rapidly degraded. Furthermore, specific miRNA-mRNA partners must be inferred bioinformatically because all miRNA-mRNA pairs co-precipitated together. Finally, SILAC directly identifies the final product of miRNA regulation, the protein itself, but is insensitive and therefore misses rare proteins and small fold changes in protein levels.

In light of the limitations with current miRNA target identification methods, we felt an additional global assay to identify functional miRNA targets by an alternative mechanism was needed. To fulfill this need, we licensed a technology invented by Joop Gaken and Azim Mohamedali of King&#x2019;s College London. Their invention is a dual selection fusion protein, specifically, a thymidine kinase-zeocin fusion, regulated by a cDNA library of potential miRNA target sequences in its 3&#x2032;UTR. Cells stably transfected with and expressing TKzeo-cDNA constructs can be selected with zeocin, as illustrated in Fig 2. After expressing a miRNA of interest, that miRNA&#x2019;s targets can be selected with ganciclovir, as illustrated in Fig 3. Ganciclovir kills cells expressing thymidine kinase, that is, any cells expressing TKzeo-cDNA lacking a target for the miRNA of interest. Targeted cDNA can be isolated by PCR amplification of DNA from cells that survive ganciclovir using primers that flank the cDNA, and PCR products can be sequenced to identify targets. 

We have developed the King&#x2019;s College technology into a new tool for global identification and discovery of functional human miRNA targets &#x2013; the MISSION Target ID Library. With the Target ID Library, users can isolate miRNA targets by a series of mammalian cell culture transfection and drug selection steps. The Library is comprehensive, and contains 66-79% of human genes. Initial results indicate that both previously discovered as well as new targets can be isolated from the MISSION Target ID Library.

Although the protocol is of some length, use of the Target ID Library requires standard molecular lab techniques &#x2013; mammalian cell culture, transfection, drug selection, PCR, and sequencing &#x2013; and therefore, should be well within the means of most biologists. We suggest that sufficient attention be paid to proper experimental design, optimizing culture conditions, and most importantly, pre-testing each new cell type to determine optimum drug levels for selection steps (kill-curves) to ensure success with the Target ID Library.

As with all miRNA target identification methods, it is highly recommended that targets identified by screening the Target ID Library be validated with a second method, such as microarray, qRT-PCR, reporter assay (i.e., luciferase), or Western analysis.

<table><tbody><tr><td>JumpStart REDTaq ReadyMix</td><td><a href="http://www.sigmaaldrich.com/catalog/product/sigma/P0982?lang=en&amp;amp;region=US&amp;amp;cm_mmc=affiliate-_-JOVE-_-video-_-target_id" target="_blank"><img src="/img/new_window.png" /> Sigma-Aldrich</a></td><td>P0982</td><td> </td></tr><tr><td>&lt;a href=&quot;http://www.sigmaaldrich.com/catalog/product/sigma/G2536?lang=en&amp;amp;region=US&amp;amp;cm_mmc=affiliate-_-JOVE-_-video-_-target_id&quot; target=&quot;_blank&quot;&gt;Ganciclovir&lt;/a&gt;</td><td>&lt;a href=&quot;http://www.sigmaaldrich.com/&quot; target=&quot;_blank&quot;&gt;Sigma-Aldrich&lt;/a&gt;</td><td>&lt;a href=&quot;http://www.sigmaaldrich.com/catalog/product/sigma/G2536?lang=en&amp;amp;region=US&amp;amp;cm_mmc=affiliate-_-JOVE-_-video-_-target_id&quot; target=&quot;_blank&quot;&gt;G2536&lt;/a&gt;</td><td> </td></tr><tr><td>G418</td><td>&lt;a href=&quot;http://www.sigmaaldrich.com/&quot; target=&quot;_blank&quot;&gt;Sigma-Aldrich&lt;/a&gt;</td><td>G8168</td><td> </td></tr><tr><td>Puromycin</td><td><a href="http://www.sigmaaldrich.com/catalog/product/sigma/P9620?lang=en&amp;amp;region=US&amp;amp;cm_mmc=affiliate-_-JOVE-_-video-_-target_id" target="_blank"><img src="/img/new_window.png" /> Sigma-Aldrich</a></td><td>P9620</td><td> </td></tr><tr><td>Topo TA</td><td>Invitrogen</td><td>KNM4500-01</td><td> </td></tr><tr><td>GenElute Mammalian Genomic DNA Miniprep Kit</td><td><a href="http://www.sigmaaldrich.com/catalog/product/sigma/g1n10?lang=en&amp;amp;region=US&amp;amp;cm_mmc=affiliate-_-JOVE-_-video-_-target_id" target="_blank"><img src="/img/new_window.png" /> Sigma-Aldrich</a></td><td>G1N10</td><td> </td></tr><tr><td>Cell Specific Nucleofection kit</td><td>Lonza Inc.</td><td></td><td> </td></tr><tr><td>Nucleofector</td><td>Lonza Inc.</td><td>AAD-1001</td><td> </td></tr><tr><td>Zeocin</td><td>Invitrogen</td><td>&lt;a href=&quot;http://products.invitrogen.com/ivgn/en/US/adirect/invitrogen?cmd=catProductDetail&amp;amp;productID=R25001&quot;&gt;R250-01&lt;/a&gt;</td><td> </td></tr><tr><td>&lt;a href=&quot;http://www.sigmaaldrich.com/catalog/product/sigma/mreh01?lang=en&amp;amp;region=US&amp;amp;cm_mmc=affiliate-_-JOVE-_-video-_-target_id&quot; target=&quot;_blank&quot;&gt;Target ID Library&lt;/a&gt;</td><td>&lt;a href=&quot;http://www.sigmaaldrich.com/&quot; target=&quot;_blank&quot;&gt;Sigma-Aldrich&lt;/a&gt;</td><td>&lt;a href=&quot;http://www.sigmaaldrich.com/catalog/product/sigma/mreh01?lang=en&amp;amp;region=US&amp;amp;cm_mmc=affiliate-_-JOVE-_-video-_-target_id&quot; target=&quot;_blank&quot;&gt;MREH01&lt;/a&gt;</td><td></td></tr></tbody></table>

genome-wide screen for mirna targets using the mission target id library

The Target ID Library is designed to assist in discovery and identification of microRNA (miRNA) targets. The Target ID Library is a plasmid-based, genome-wide cDNA library cloned into the 3'UTR downstream from the dual-selection fusion protein, thymidine kinase-zeocin (TKzeo). The first round of selection is for stable transformants, followed with introduction of a miRNA of interest, and finally, selecting for cDNAs containing the miRNA's target. Selected cDNAs are identified by sequencing (see Figure 1-3 for Target ID Library Workflow and details).
To ensure broad coverage of the human transcriptome, Target ID Library cDNAs were generated via oligo-dT priming using a pool of total RNA prepared from multiple human tissues and cell lines. Resulting cDNA range from 0.5 to 4 kb, with an average size of 1.2 kb, and were cloned into the p3&#900;TKzeo dual-selection plasmid (see Figure 4 for plasmid map). The gene targets represented in the library can be found on the Sigma-Aldrich webpage. Results from Illumina sequencing (Table 3), show that the library includes 16,922 of the 21,518 unique genes in UCSC RefGene (79%), or 14,000 genes with 10 or more reads (66%).

Watch this Scientific Journal Video about Genome-wide Screen for miRNA Targets Using the MISSION Target ID Library at JoVE.com

Genome-wide Screen for miRNA Targets Using the MISSION Target ID Library

The Target ID Library is a plasmid-based, genome-wide collection of cloned cDNA used to identify miRNA targets. Here we demonstrate its use and application.

The Target ID Library is designed to assist in discovery and identification of microRNA (miRNA) targets. The Target ID Library is a plasmid-based, ...

genome-wide-screen-for-mirna-targets-using-mission-target-id

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

17.5K Views.  Sigma-Aldrich. The Target ID Library is a plasmid-based, genome-wide collection of cloned cDNA used to identify miRNA targets. Here we demonstrate its use and application.

Video: Genome-wide Screen for miRNA Targets Using the MISSION Target ID Library

Biotin-based Pulldown Assay to Validate mRNA Targets of Cellular miRNAs

MicroRNAs (miRNAs) are a class of small noncoding RNAs that post-transcriptionally regulate cellular gene expression. MiRNAs bind to the 3&#39; untranslated region (UTR) of target mRNA to inhibit protein translation or in some instances cause mRNA degradation. The binding of the miRNA to the 3&#39; UTR of the target mRNA is mediated by a 2&#8211;8 nucleotide seed sequence at the 5&#39; end of miRNA. While the role of miRNAs as cellular regulatory molecules is well established, identification of the target mRNAs with functional relevance remains a challenge. Bioinformatic tools have been employed to predict sequences within the 3&#39; UTR of mRNAs as potential targets for miRNA binding. These tools have also been utilized to determine the evolutionary conservation of such sequences among related species in an attempt to predict functional role. However, these computational methods often generate false positive results and are limited to predicting canonical interaction between miRNA and mRNA. Therefore, experimental procedures that measure direct binding of miRNA to its mRNA target are necessary to establish functional interaction. In this report, we describe a sensitive method for validating direct interaction between the cellular miRNA miR-125b and the 3&#39; UTR of PARP-1 mRNA. We elaborate a protocol in which synthetic biotinylated-miRNA mimics were transfected into mammalian cells and the miRNA-mRNA complex in the cellular lysate was pulled down with streptavidin-coated magnetic beads. Finally, the target mRNA in the pulled-down nucleic acid complex was quantified using a qPCR-based strategy.

This report describes a fast and reliable method for validating mRNA targets of cellular miRNAs. The method uses synthetic biotinylated Locked Nucleic Acid (LNA)-based miRNA mimics to capture target mRNA. Subsequently, streptavidin-coated magnetic beads are employed to pulldown the target mRNA for quantification by qPCR polymerase chain reaction.

MicroRNAs (miRNAs) are a class of small noncoding RNAs that post-transcriptionally regulate cellular gene expression. MiRNAs bind to the 3&#39; ...

Genetics

Detection of a Circulating MicroRNA Custom Panel in Patients with Metastatic Colorectal Cancer

There is increasing interest in liquid biopsy for cancer diagnosis, prognosis and therapeutic monitoring, creating the need for reliable and useful biomarkers for clinical practice. Here, we present a protocol to extract, reverse transcribe and evaluate the expression levels of circulating miRNAs from plasma samples of patients with colorectal cancer (CRC). microRNAs (miRNAs) are a class of non-coding RNAs of 18-25 nucleotides in length that regulate the expression of target genes at the translational level and play an important role, including that of pro- and anti-angiogenic function, in the physiopathology of various organs. miRNAs are stable in biological fluids such as serum and plasma, which renders them ideal circulating biomarkers for cancer diagnosis, prognosis and treatment decision-making and monitoring. Circulating miRNA extraction was performed using a rapid and effective method that involves both organic and column-based methodologies. For miRNA retrotranscription, we used a multi-step procedure that considers polyadenylation at 3&#39; and the ligation of an adapter at 5&#39; of the mature miRNAs, followed by random miRNA pre-amplification. We selected a 24-miRNA custom panel to be tested by quantitative real-time polymerase chain reaction (qRT-PCR) and spotted the miRNA probes on array custom plates. We performed qRT-PCR plate runs on a real-time PCR System. Housekeeping miRNAs for normalization were selected using GeNorm software (v. 3.2). Data were analyzed using Expression suite software (v 1.1) and statistical analyses were performed. The method proved to be reliable and technically robust and could be useful to evaluate biomarker levels in liquid samples such as plasma and/or serum.

We present a protocol to evaluate the expression levels of circulating microRNA (miRNAs) in plasma samples from cancer patients. In particular, we used a commercially available kit for circulating miRNA extraction and reverse transcription. Finally, we analyzed a panel of 24 selected miRNAs using real-time pre-spotted probe custom plates.

There is increasing interest in liquid biopsy for cancer diagnosis, prognosis and therapeutic monitoring, creating the need for reliable and useful ...

Cancer Research

Characterization of Functionally Associated miRNAs in Glioblastoma and their Engineering into Artificial Clusters for Gene Therapy

The biological relevance of microRNAs (miRNAs) in health and disease significantly relies on specific combinations of many simultaneously deregulated miRNAs rather than the action of a single miRNA. The characterization of these specific miRNAs modules is a fundamental step in maximizing their use in therapy. This is extremely relevant because their combinatorial attributes can be practically exploited. Described here is a method to define a specific miRNA signature relevant to the control of oncogenic chromatin repressors in glioblastoma. The approach first defines a general group of miRNAs that are deregulated in tumors in comparison to normal tissue. The analysis is further refined by differential culture conditions, underscoring a subgroup of miRNAs that are co-expressed simultaneously during specific cellular states. Finally, the miRNAs that satisfy these filters are combined into an artificial polycistronic transgenes, which is based on a scaffold of naturally existing miRNA clusters genes, then used for overexpression of these miRNA modules into receiving cells.

Described here is a protocol for characterizing modules of biologically synergistic miRNAs and their assembly into short transgenes, which allows simultaneous overexpression for gene therapy applications.

The biological relevance of microRNAs (miRNAs) in health and disease significantly relies on specific combinations of many simultaneously deregulated ...

Genome-Scale Screening to Identify and Compare Therapeutic Targets in Tumors

Genetic Profiling and Genome-Scale Dropout Screening to Identify Therapeutic Targets in Mouse Models of Malignant Peripheral Nerve Sheath Tumor

Malignant Peripheral Nerve Sheath Tumors (MPNSTs) are derived from Schwann cells or their precursors. In patients with the tumor susceptibility syndrome neurofibromatosis type 1 (NF1), MPNSTs are the most common malignancy and the leading cause of death. These rare and aggressive soft-tissue sarcomas offer a stark future, with 5-year disease-free survival rates of 34-60%. Treatment options for individuals with MPNSTs are disappointingly limited, with disfiguring surgery being the foremost treatment option. Many once-promising therapies such as tipifarnib, an inhibitor of Ras signaling, have failed clinically. Likewise, phase II clinical trials with erlotinib, which targets the epidermal growth factor (EFGR), and sorafenib, which targets the vascular endothelial growth factor receptor (VEGF), platelet-derived growth factor receptor (PDGF), and Raf, in combination with standard chemotherapy, have also failed to produce a response in patients. 
In recent years, functional genomic screening methods combined with genetic profiling of cancer cell lines have proven useful for identifying essential cytoplasmic signaling pathways and the development of target-specific therapies. In the case of rare tumor types, a variation of this approach known as cross-species comparative oncogenomics is increasingly being used to identify novel therapeutic targets. In cross-species comparative oncogenomics, genetic profiling and functional genomics are performed in genetically engineered mouse (GEM) models and the results are then validated in the rare human specimens and cell lines that are available. 
This paper describes how to identify candidate driver gene mutations in human and mouse MPNST cells using whole exome sequencing (WES). We then describe how to perform genome-scale shRNA screens to identify and compare critical signaling pathways in mouse and human MPNST cells and identify druggable targets in these pathways. These methodologies provide an effective approach to identifying new therapeutic targets in a variety of human cancer types.

Author Spotlight: Finding New Therapeutic Targets for Malignant Peripheral Nerve Sheath Tumor Through Genome-Scale shRNA Screens 

We have developed a cross-species comparative oncogenomics approach utilizing genomic analyses and functional genomic screens to identify and compare therapeutic targets in tumors arising in genetically engineered mouse models and the corresponding human tumor type.

Malignant Peripheral Nerve Sheath Tumors (MPNSTs) are derived from Schwann cells or their precursors. In patients with the tumor susceptibility syndrome ...

MISSION esiRNA for RNAi Screening in Mammalian Cells

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as small interfering RNAs (siRNAs). The short double-stranded RNAs originate from longer double stranded precursors by the activity of Dicer, a protein of the RNase III family of endonucleases. The resulting fragments are components of the RNA-induced silencing complex (RISC), directing it to the cognate target mRNA. RISC cleaves the target mRNA thereby reducing the expression of the encoded protein1,2,3. RNAi has become a powerful and widely used experimental method for loss of gene function studies in mammalian cells utilizing small interfering RNAs.
Currently two main methods are available for the production of small interfering RNAs. One method involves chemical synthesis, whereas an alternative method employs endonucleolytic cleavage of target specific long double-stranded RNAs by RNase III in vitro. Thereby, a diverse pool of siRNA-like oligonucleotides is produced which is also known as endoribonuclease-prepared siRNA or esiRNA. A comparison of efficacy of chemically derived siRNAs and esiRNAs shows that both triggers are potent in target-gene silencing. Differences can, however, be seen when comparing specificity. Many single chemically synthesized siRNAs produce prominent off-target effects, whereas the complex mixture inherent in esiRNAs leads to a more specific knockdown10.
In this study, we present the design of genome-scale MISSION esiRNA libraries and its utilization for RNAi screening exemplified by a DNA-content screen for the identification of genes involved in cell cycle progression. We show how to optimize the transfection protocol and the assay for screening in high throughput. We also demonstrate how large data-sets can be evaluated statistically and present methods to validate primary hits. Finally, we give potential starting points for further functional characterizations of validated hits.

Here we use a human esiRNA library in a high-throughput screen for genes involved in cell division. We demonstrate how to set up and conduct an esiRNA screens, as well as how to analyze and validate the results.

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as ...

Genome-wide Analysis of HDAC Inhibitor-mediated Modulation of microRNAs and mRNAs in B Cells Induced to Undergo Class-switch DNA Recombination and Plasma Cell Differentiation

Antibody responses are accomplished through several critical B cell-intrinsic processes, including somatic hypermutation (SHM), class-switch DNA recombination (CSR), and plasma cell differentiation. In recent years, epigenetic modifications or factors, such as histone deacetylation and microRNAs (miRNAs), have been shown to interact with B-cell genetic programs to shape antibody responses, while the dysfunction of epigenetic factors has been found to lead to autoantibody responses. Analyzing genome-wide miRNA and mRNA expression in B cells in response to epigenetic modulators is important for understanding the epigenetic regulation of B-cell function and antibody response. Here, we demonstrate a protocol for inducing B cells to undergo CSR and plasma cell differentiation, treating these B cells with histone deacetylase (HDAC) inhibitors (HDIs), and analyzing mRNA and microRNA expression. In this protocol, we directly analyze complementary DNA (cDNA) sequences using next-generation mRNA sequencing (mRNA-seq) and miRNA-seq technologies, mapping of the sequencing reads to the genome, and quantitative reverse transcription (qRT)-PCR. With these approaches, we have defined that, in B cells induced to undergo CSR and plasma cell differentiation, HDI, an epigenetic regulator, selectively modulates miRNA and mRNA expression and alters CSR and plasma cell differentiation.

Epigenetic factors can interact with genetic programs to modulate gene expression and regulate B cell function. By combining in vitro B-cell stimulation, qRT-PCR, and high-throughput microRNA-sequence and mRNA-sequence approaches, we can analyze the epigenetic modulation of miRNA and gene expression in B cells.

Antibody responses are accomplished through several critical B cell-intrinsic processes, including somatic hypermutation (SHM), class-switch DNA ...

Immunology and Infection

MISSION LentiPlex Pooled shRNA Library Screening in Mammalian Cells

RNA interference (RNAi) is an intrinsic cellular mechanism for the regulation of gene expression. Harnessing the innate power of this system enables us to knockdown gene expression levels in loss of gene function studies.
There are two main methods for performing RNAi. The first is the use of small interfering RNAs (siRNAs) that are chemically synthesized, and the second utilizes short-hairpin RNAs (shRNAs) encoded within plasmids 1. The latter can be transfected into cells directly or packaged into replication incompetent lentiviral particles. The main advantages of using lentiviral shRNAs is the ease of introduction into a wide variety of cell types, their ability to stably integrate into the genome for long term gene knockdown and selection, and their efficacy in conducting high-throughput loss of function screens. To facilitate this we have created the LentiPlex pooled shRNA library.
The MISSION LentiPlex Human shRNA Pooled Library is a genome-wide lentiviral pool produced using a proprietary process. The library consists of over 75,000 shRNA constructs from the TRC collection targeting 15,000+ human genes 2. Each library is tested for shRNA representation before product release to ensure robust library coverage. The library is provided in a ready-to-use lentiviral format at titers of at least 5 x 108 TU/ml via p24 assay and is pre-divided into ten subpools of approximately 8,000 shRNA constructs each. Amplification and sequencing primers are also provided for downstream target identification.
Previous studies established a synergistic antitumor activity of TRAIL when combined with Paclitaxel in A549 cells, a human lung carcinoma cell line 3, 4. In this study we demonstrate the application of a pooled LentiPlex shRNA library to rapidly conduct a positive selection screen for genes involved in the cytotoxicity of A549 cells when exposed to TRAIL and Paclitaxel. One barrier often encountered with high-throughput screens is the cost and difficulty in deconvolution; we also detail a cost-effective polyclonal approach utilizing traditional sequencing.

Here we use a human LentiPlex pooled library and traditional sequencing methods to identify gene targets promoting cell survival. We demonstrate how to set up and deconvolute a LentiPlex screen and validate the results.

RNA interference (RNAi) is an intrinsic cellular mechanism for the regulation of gene expression. Harnessing the innate power of this system enables us to ...

Identifying Targets of Human microRNAs with the LightSwitch Luciferase Assay System using 3'UTR-reporter Constructs and a microRNA Mimic in Adherent Cells

MicroRNAs (miRNAs) are important regulators of gene expression and play a role in many biological processes. More than 700 human miRNAs have been identified so far with each having up to hundreds of unique target mRNAs. Computational tools, expression and proteomics assays, and chromatin-immunoprecipitation-based techniques provide important clues for identifying mRNAs that are direct targets of a particular miRNA. In addition, 3'UTR-reporter assays have become an important component of thorough miRNA target studies because they provide functional evidence for and quantitate the effects of specific miRNA-3'UTR interactions in a cell-based system. To enable more researchers to leverage 3'UTR-reporter assays and to support the scale-up of such assays to high-throughput levels, we have created a genome-wide collection of human 3'UTR luciferase reporters in the highly-optimized LightSwitch Luciferase Assay System. The system also includes synthetic miRNA target reporter constructs for use as positive controls, various endogenous 3'UTR reporter constructs, and a series of standardized experimental protocols.
Here we describe a method for co-transfection of individual 3'UTR-reporter constructs along with a miRNA mimic that is efficient, reproducible, and amenable to high-throughput analysis.

MicroRNAs (miRNAs) are important regulators of gene expression and have been shown to play a role in numerous biological processes. To better understand miRNA-UTR interactions, we have created a genome-wide collection of 3 UTR luciferase reporters paired with a novel luciferase gene and assay reagent, the LightSwitch system.

MicroRNAs (miRNAs) are important regulators of gene expression and play a role in many biological processes.  More than 700 human miRNAs have been ...

Lung microRNA Profiling Across the Estrous Cycle in Ozone-exposed Mice

MicroRNA (miRNA) profiling has become of interest to researchers working in various research areas of biology and medicine. Current studies show a promising future of using miRNAs in the diagnosis and care of lung diseases. Here, we define a protocol for miRNA profiling to measure the relative abundance of a group of miRNAs predicted to regulate inflammatory genes in the lung tissue from of an ozone-induced airway inflammation mouse model. Because it has been shown that circulating sex hormone levels can affect the regulation of lung innate immunity in females, the purpose of this method is to describe an inflammatory miRNA profiling protocol in female mice, taking into consideration the estrous cycle stage of each animal at the time of ozone exposure. We also address applicable bioinformatics approaches to miRNA discovery and target identification methods using limma, an R/Bioconductor software, and functional analysis software to understand the biological context and pathways associated with differential miRNA expression.

Here we describe a method to assess lung expression of miRNAs that are predicted to regulate inflammatory genes using mice exposed to ozone or filtered air at different stages of the estrous cycle.

MicroRNA (miRNA) profiling has become of interest to researchers working in various research areas of biology and medicine. Current studies show a ...

Detection of miRNA Targets in High-throughput Using the 3'LIFE Assay

Luminescent Identification of Functional Elements in 3&#8217;UTRs (3&#8217;LIFE) allows the rapid identification of targets of specific miRNAs within an array of hundreds of queried 3&#8217;UTRs. Target identification is based on the dual-luciferase assay, which detects binding at the mRNA level by measuring translational output, giving a functional readout of miRNA targeting. 3&#8217;LIFE uses non-proprietary buffers and reagents, and publically available reporter libraries, making genome-wide screens feasible and cost-effective. 3&#8217;LIFE can be performed either in a standard lab setting or scaled up using liquid handling robots and other high-throughput instrumentation. We illustrate the approach using a dataset of human 3&#8217;UTRs cloned in 96-well plates, and two test miRNAs, let-7c and miR-10b. We demonstrate how to perform DNA preparation, transfection, cell culture and luciferase assays in 96-well format, and provide tools for data analysis. In conclusion 3&#39;LIFE is highly reproducible, rapid, systematic, and identifies high confidence targets.

Luminescent identification of functional elements in 3&#8217; untranslated regions (3&#8217;UTRs) (3&#8217;LIFE) is a technique to identify functional regulation in 3&#8217;UTRs by miRNAs or other regulatory factors. This protocol utilizes high-throughput methodology such as 96-well transfection and luciferase assays to screen hundreds of putative interactions for functional repression.

Luminescent Identification of Functional Elements in 3&#8217;UTRs (3&#8217;LIFE) allows the rapid identification of targets of specific miRNAs within an ...