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 Joop G&auml;ken of King&#x2019;s College London for freely sharing unpublished results and suggestions, and Qazi Hamid of RxBiosciences for preparing a great batch of cDNA and his persistence in getting it cloned.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.

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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. 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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. 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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 border="1" >
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td><a href="http://www.sigmaaldrich.com/catalog/product/sigma/P0982?lang=en&region=US&cm_mmc=affiliate-_-JOVE-_-video-_-target_id" target="_blank">JumpStart
 REDTaq 
 ReadyMix</a></td>
 <td><a href="http://www.sigmaaldrich.com/" target="_blank">Sigma-Aldrich</a></td>
 <td><a href="http://www.sigmaaldrich.com/catalog/product/sigma/P0982?lang=en&region=US&cm_mmc=affiliate-_-JOVE-_-video-_-target_id" target="_blank">P0982</a></td>
 <td>&nbsp;
 </td>
 </tr>
 <tr>
 <td><a href="http://www.sigmaaldrich.com/catalog/product/sigma/G2536?lang=en&region=US&cm_mmc=affiliate-_-JOVE-_-video-_-target_id" target="_blank">Ganciclovir</a></td>
 <td><a href="http://www.sigmaaldrich.com/" target="_blank">Sigma-Aldrich</a></td>
 <td><a href="http://www.sigmaaldrich.com/catalog/product/sigma/G2536?lang=en&region=US&cm_mmc=affiliate-_-JOVE-_-video-_-target_id" target="_blank">G2536</a></td>
 <td>&nbsp;
 </td>
 </tr>
 <tr>
 <td>G418</td>
 <td><a href="http://www.sigmaaldrich.com/" target="_blank">Sigma-Aldrich</a></td>
 <td>G8168</td>
 <td>&nbsp;
 </td>
 </tr>
 <tr>
 <td><a href="http://www.sigmaaldrich.com/catalog/product/sigma/P9620?lang=en&region=US&cm_mmc=affiliate-_-JOVE-_-video-_-target_id" target="_blank">Puromycin</a></td>
 <td><a href="http://www.sigmaaldrich.com/" target="_blank">Sigma-Aldrich</a></td>
 <td><a href="http://www.sigmaaldrich.com/catalog/product/sigma/P9620?lang=en&region=US&cm_mmc=affiliate-_-JOVE-_-video-_-target_id" target="_blank">P9620</a></td>
 <td>&nbsp;
 </td>
 </tr>
 <tr>
 <td>Topo TA</td>
 <td>Invitrogen</td>
 <td>KNM4500-01
 &nbsp;
 </td>
 <td>&nbsp;
 </td>
 </tr>
 <tr>
 <td><a href="http://www.sigmaaldrich.com/catalog/product/sigma/g1n10?lang=en&region=US&cm_mmc=affiliate-_-JOVE-_-video-_-target_id" target="_blank">GenElute Mammalian Genomic DNA Miniprep Kit</a></td>
 <td><a href="http://www.sigmaaldrich.com/" target="_blank">Sigma-Aldrich</a></td>
 <td><a href="http://www.sigmaaldrich.com/catalog/product/sigma/g1n10?lang=en&region=US&cm_mmc=affiliate-_-JOVE-_-video-_-target_id" target="_blank">G1N10</a></td>
 <td>&nbsp;
 </td>
 </tr>
 <tr>
 <td>Cell Specific Nucleofection kit</td>
 <td>Lonza</td>
 <td>&nbsp;
 </td>
 <td>&nbsp;
 </td>
 </tr>
 <tr>
 <td>Nucleofector</td>
 <td>Lonza</td>
 <td>AAD-1001</td>
 <td>&nbsp;
 </td>
 </tr>
 <tr>
 <td>Zeocin</td>
 <td>Invitrogen</td>
 <td><a href="http://products.invitrogen.com/ivgn/en/US/adirect/invitrogen?cmd=catProductDetail&amp;productID=R25001">R250-01</a></td>
 <td>&nbsp;
 </td>
 </tr>
 <tr>
 <td height="1"><a href="http://www.sigmaaldrich.com/catalog/product/sigma/mreh01?lang=en&region=US&cm_mmc=affiliate-_-JOVE-_-video-_-target_id" target="_blank">Target ID Library</a></td>
 <td><a href="http://www.sigmaaldrich.com/" target="_blank">Sigma-Aldrich</a></td>
 <td><a href="http://www.sigmaaldrich.com/catalog/product/sigma/mreh01?lang=en&region=US&cm_mmc=affiliate-_-JOVE-_-video-_-target_id" target="_blank">MREH01</a></td>
 <td>&nbsp;</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, ...

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17.4K 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.