micro RNA and qRT-PCR

Strategies to determine the biological function of microRNAs.
NATURE GENETICS SUPPLEMENT VOLUME 38 JUNE 2006
Jan Krützfeldt, Matthew N Poy & Markus Stoffel

MicroRNAs (miRNAs) are regulators of gene expression that control many biological processes in development, differentiation, growth and metabolism. Their expression levels, small size, abundance of repetitive copies in the genome and mode of action pose unique challenges in studies elucidating the function of miRNAs. New technologies for identification, expression profiling and target gene validation, as well as manipulation of miRNA expression in vivo, will facilitate the study of their contribution to biological processes and disease. Such information will be crucial to exploit the emerging knowledge of miRNAs for the development of new human therapeutic applications.

Genomics of microRNA.
TRENDS in Genetics Vol.22 No.3 March 2006
V. Narry Kim1 and Jin-Wu Nam2
1Department of Biological Sciences and Research Center for Functional Cellulomics, Seoul National University, Korea
2Graduate Program in Bioinformatics, Seoul National University, Seoul, 151-742, Korea

Discovered just over a decade ago, microRNA (miRNA) is now recognized as one of the major regulatory gene families in eukaryotic cells. Hundreds of miRNAs have been found in animals, plants and viruses, and there are certainly more to come. Through specific base-pairing with mRNAs, these tiny w22-nt RNAs induce mRNA degradation or translational repression, or both. Because a miRNA can target numerous mRNAs, often in combination with other miRNAs, miRNAs operate highly complex regulatory networks. In this article, we summarize the current status of miRNA gene mining and miRNA expression profiling. We also review up-todate knowledge of miRNA gene structure and the biogenesis mechanism. Our focus is on animal miRNAs.

Tracing microRNA patterns in mice.
NATURE GENETICS VOLUME 36 | NUMBER 10 | OCTOBER 2004
John Cobb & Denis Duboule

MicroRNAs are important in the development of worms, flies and plants. A new study shows how the temporal and spatial expression patterns of such RNAs can be determined in mouse embryos, providing insights into the emerging role of microRNAs in Hox gene regulation.

Target mimics modulate miRNAs.
NATURE GENETICS | VOLUME 39 | NUMBER 8 | AUGUST 2007
Daniel H Chitwood & Marja C P Timmermans

A 23-nucleotide sequence conserved in species from rice to Arabidopsis thaliana in a family of noncoding RNAs resembles a cleavable miRNA target site—but not exactly. A new study demonstrates that the site is not cleaved and instead negatively regulates miRNA activity through mimicry.

Target mimicry provides a new mechanism for regulation of microRNA activity.
NATURE GENETICS VOLUME 39 [ NUMBER 8 [ AUGUST 2007
Jose´ Manuel Franco-Zorrilla1, Adria´n Valli1, Marco Todesco2, Isabel Mateos1, Marı´a Isabel Puga1,
Ignacio Rubio-Somoza2, Antonio Leyva1, Detlef Weigel2, Juan Antonio Garcı´a1 & Javier Paz-Ares1

MicroRNAs (miRNA) regulate key aspects of development and physiology in animals and plants. These regulatory RNAs act as guides of effector complexes to recognize specific mRNA sequences based on sequence complementarity, resulting in translational repression or site-specific cleavage1,2. In plants, most miRNA targets are cleaved and show almost perfect complementarity with the miRNAs around the cleavage site3–8. Here, we examined the non–protein coding gene IPS1 (INDUCED BY PHOSPHATE STARVATION1) from Arabidopsis thaliana. IPS1 contains a motif with sequence complementarity to the phosphate (Pi) starvation–induced miRNA miR-399, but the pairing is interrupted by a mismatched loop at the expected miRNA cleavage site. We show that IPS1 RNA is not cleaved but instead sequesters miR-399. Thus, IPS1 overexpression results in increased accumulation of the miR-399 target PHO2 mRNA and, concomitantly, in reduced shoot Pi content5–8. Engineering of IPS1 to be cleavable abolishes its inhibitory activity on miR-399. We coin the term ‘target mimicry’ to define this mechanism of inhibition of miRNA activity. Target mimicry can be generalized beyond the control of Pi homeostasis, as demonstrated using artificial target mimics.

Lessons from Nature: microRNA-based shRNA libraries.
NATURE METHODS | VOL.3 NO.9 | SEPTEMBER 2006 | 707
Kenneth Chang1, Stephen J Elledge2 & Gregory J Hannon1

Loss-of-function genetics has proven essential for interrogating the functions of genes and for probing their roles within the complex circuitry of biological pathways. In many systems, technologies allowing the use of such approaches were lacking before the discovery of RNA interference (RNAi). We have constructed first-generation short hairpin RNA (shRNA) libraries modeled after precursor microRNAs (miRNAs) and secondgeneration libraries modeled after primary miRNA transcripts (the Hannon-Elledge libraries). These libraries were arrayed, sequence-verified, and cover a substantial portion of all known and predicted genes in the human and mouse genomes. Comparison of first- and second-generation libraries indicates that RNAi triggers that enter the RNAi pathway through a more natural route yield more effective silencing. These large-scale resources are functionally versatile, as they can be used in transient and stable studies, and for constitutive or inducible silencing. Library cassettes can be easily shuttled into vectors that contain different promoters and/or that provide different modes of viral delivery.

Identification of Differentially Expressed MicroRNAs by Microarray:
A Possible Role for MicroRNA Genes in Pituitary Adenomas
J. Cell. Physiol. 210: 370–377, 2007
ARIANNA BOTTONI,1 MARIA CHIARA ZATELLI,1 MANUELA FERRACIN,2 FEDERICO TAGLIATI,1
DANIELA PICCIN,1 CRISTINA VIGNALI,1 GEORGE A. CALIN,3 MASSIMO NEGRINI,2 CARLO M. CROCE,3
AND ETTORE C. DEGLI UBERTI1*
1Department of Biomedical Sciences and Advanced Therapies, Section of Endocrinology, University of Ferrara, Ferrara, Italy
2Department of Experimental and Diagnostic Medicine and Interdepartment Center for Cancer Research, University of Ferrara
3Department of Molecular Virology, Immunology, and Medical Genetics and Cancer Comprehensive Center, Columbus, Ohio

MicroRNAs (miRNAs) are small non-coding RNAs that control gene expression by targeting mRNA. It has been demonstrated that miRNA expression is altered in many human cancers, suggesting that they may play a role in human neoplasia. To determine whether miRNA expression is altered in pituitary adenomas, we analyzed the entire miRNAome in 32 pituitary adenomas and in 6 normal pituitary samples by microarray and by Real-Time PCR. Here, we show that 30 miRNAs are differentially expressed between normal pituitary and pituitary adenomas. Moreover, 24 miRNAs were identified as a predictive signature of pituitary adenoma and 29 miRNAs were able to predict pituitary adenoma histotype. miRNA expression could differentiate micro- from macro-adenomas and treated from non-treated patient samples. Several of the identified miRNAs are involved in cell proliferation and apoptosis, suggesting that their deregulated expression may be involved in pituitary tumorigenesis. Predictive miRNAs could be potentially useful diagnostic markers, improving the classification of pituitary adenomas.

RNA polymerase III transcribes human microRNAs.
NATURE STRUCTURAL & MOLECULAR BIOLOGY VOLUME 13 NUMBER 12 DECEMBER 2006
Glen M Borchert1,2, William Lanier2,3 & Beverly L Davidson1,4,5

Prior work demonstrates that mammalian microRNA (miRNA or miR) expression requires RNA polymerase II (Pol II). However, the transcriptional requirements of many miRNAs remain untested. Our genomic analysis of miRNAs in the human chromosome 19 miRNA cluster (C19MC) revealed that they are interspersed among Alu repeats. Because Alu transcription occurs through RNA Pol III recruitment, and we found that Alu elements upstream of C19MC miRNAs retain sequences important for Pol III activity, we tested the promoter requirements of C19MC miRNAs. Chromatin immunoprecipitation and cell-free transcription assays showed that Pol III, but not Pol II, is associated with miRNA genomic sequence and sufficient for transcription. Moreover, the mature miRNA sequences of approximately 50 additional human miRNAs lie within Alu and other known repetitive elements. These findings extend the current view of miRNA origins and the transcriptional machinery driving their expression.

Identification by Real-time PCR of 13 mature microRNAs differentially expressed in colorectal cancer and non-tumoral tissues.
Molecular Cancer 2006, 5:29
E Bandrés*1, E Cubedo1, X Agirre2, R Malumbres1, R Zárate1, N Ramirez1,
A Abajo1, A Navarro3, I Moreno4, M Monzó3 and J García-Foncillas1

MicroRNAs (miRNAs) are short non-coding RNA molecules playing regulatory roles by repressing translation or cleaving RNA transcripts. Although the number of verified human miRNA is still expanding, only few have been functionally described. However, emerging evidences suggest the potential involvement of altered regulation of miRNA in pathogenesis of cancers and these genes are thought to function as both tumours suppressor and oncogenes. In our study, we examined by Real-Time PCR the expression of 156 mature miRNA in colorectal cancer. The analysis by several bioinformatics algorithms of colorectal tumours and adjacent nonneoplastic tissues from patients and colorectal cancer cell lines allowed identifying a group of 13 miRNA whose expression is significantly altered in this tumor. The most significantly deregulated miRNA being miR-31, miR-96, miR-133b, miR-135b, miR-145, and miR-183. In addition, the expression level of miR-31 was correlated with the stage of CRC tumor. Our results suggest that miRNA expression profile could have relevance to the biological and clinical behavior of colorectal neoplasia.

Rational Probe Optimization and Enhanced Detection Strategy for MicroRNAs Using Microarrays.
RNA Biology 2:3, 93-100
Loyal A. Goff1, Maocheng Yang2, Jessica Bowers3, Robert C. Getts3, Richard W. Padgett2, Ronald P. Hart1,*
1W.M. Keck Center for Collaborative Neuroscience; 2Waksman Institute;
Department of Molecular Biology and Biochemistry; and Cancer Center of New
Jersey; Rutgers University; Piscataway, New Jersey USA
3Genisphere Inc..; Hatfield, Pennsylvania USA

MicroRNAs (miRNAs) are post-transcriptional regulators participating in biological processes ranging from differentiation to carcinogenesis. We developed a rational probe design algorithm and a sensitive labelling scheme for optimizing miRNA microarrays. Our microarray contains probes for all validated miRNAs from five species, with the potential for drawing on species conservation to identify novel miRNAs with homologous probes. These methods are useful for high-throughput analysis of micro RNAs from various sources, and allow analysis with limiting quantities of RNA. The system design can also be extended for use on Luminex beads or on 96-well plates in an ELISA-style assay. We optimized hybridization temperatures using sequence variations on 20 of the probes and determined that all probes distinguish wild-type from 2 nt mutations, and most probes distinguish a 1 nt mutation, producing good selectivity between closely-related small RNA sequences. Results of tissue comparisons on our microarrays reveal patterns of hybridization that agree with results from Northern blots and other methods.

Potential mRNA Degradation Targets of hsa-miR-200c, Identified Using Informatics and qRT-PCR.
Cell Cycle 5:17, 1951-1956
Gregory J. Hurteau1, Simon D. Spivack2, Graham J. Brock1
1Ordway Research Institute; Albany, New York USA
2Human Toxicology & Molecular Epidemiology; Wadsworth Center; NYS Department of Health; Albany, New York USA

Using an anchored oligo(dT) based RT-PCR approach we quantified endogenous expression of ten microRNAs in six cell lines. This identified a miRNA, miR-200c, with variable expression, ranging from undetectable in MDA-MB-231 and HT1080 to highly expressed in MCF7. The variable expression provided a model system to investigate endogenous interactions between miRNAs and their computationally predicted targets. As the expression level of the predicted mRNA targets and miR-200c in these lines should have an inverse relationship if cleavage or degradation results from the interaction. To select targets for analysis we used Affymetrix expression data and computational prediction programs. Affymetrix data indicated ~3500 candidate mRNAs, absent in MCF7 and present in MDA-MB-231 or HT1080. These targets were cross-referenced against ~600 computationally predicted miR-200c targets, identifying twenty potential mRNAs. Expression analysis by qRT-PCR of these targets and an additional ten mRNAs (selected using the prediction program ranking alone) revealed four mRNAs, BIN1, TCF8, RND3 and LHFP with an inverse relationship to miR-200c. Of the remainder, the majority did not appear to be degraded (and may be translational targets) or were undetectable in the cell lines examined. Finally, inhibition of miR-200c using an anti-miRNA 2'-0-Methyl oligonucleotide (AMO) resulted in an increase in expression of one of the targets, the transcription factor TCF8. These results indicate that a single miRNA could directly affect the mRNA levels of an important transcription factor, albeit in a manner specific to cell lines. Further investigation is required to confirm this in vivo and determine any translational effects.

Real-time expression profiling of microRNA precursors in human cancer cell lines.
Nucleic Acids Research, 2005, Vol. 33, No. 17
Jinmai Jiang1, Eun Joo Lee1, Yuriy Gusev2 and Thomas D. Schmittgen1,*
1College of Pharmacy, Ohio State University, Columbus, OH, USA and 2Department of Surgery,
University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA

Our previous study described a real-timePCR method to quantify microRNA (miRNA) precursors using SYBR green detection [T. D. Schmittgen, J. Jiang, Q. Liu and L. Yang (2004) Nucleic Acids Res., 32, e43]. The present study adapted the assay to a 384-
well format and expanded it to include primers to 222 human miRNAprecursors. TaqMan minor groove binder probes were used to discriminate nearly identical members of the let-7 family of miRNA isoforms. The miRNA precursor expression was profiled in 32 human cell lines from lung, breast, colorectal, hematologic, prostate, pancreatic, and head and neck cancers. Some miRNA precursors were expressed at similar levels in many of the cell lines, while others were differentially expressed. Clustering analysis of the miRNA precursor expression data revealed that most of the cell lines clustered into their respective tissues from which each cell line was ostensibly derived. miRNA precursor expression by PCR paralleled the mature miRNA expression by northern blotting for most of the conditions studied. Our study provides PCR primer sequences to all of the known human miRNA precursors as of December 2004 and provides a database of the miRNA precursor expression inmany commonly usedhuman cancer cell lines.

Multiplexing RT-PCR for the detection of multiple miRNA species in small samples.
Biochemical and Biophysical Research Communications 343 (2006) 85–89
Kaiqin Lao *, Nan Lan Xu, Vivian Yeung, Caifu Chen, Kenneth J. Livak, Neil A. Straus*
Applied Biosystems, 850 Lincoln Centre Dr., Foster City, CA 94404, USA

MicroRNAs are short (22 nucleotides), non-coding RNAs that play critical roles in gene regulation and may be used as rapid precise diagnostic indicators of early stages of cancer. The small size of these RNAs makes detection of multiple microRNA species in very small samples problematic. Here we investigate the parameters associated with multiplexing RT-PCR to obtain relative abundance proﬁles of multiple microRNAs in small sample sizes down to the amount of RNA found in a single cell.

An oligonucleotide microchip for genome-wide microRNA profiling in human and mouse tissues.
PNAS June 29, 2004 vol. 101 no. 26, 9740–9744
Chang-Gong Liu, George Adrian Calin, Brian Meloon, Nir Gamliel, Cinzia Sevignani, Manuela Ferracin,
Calin Dan Dumitru, Masayoshi Shimizu, Simona Zupo, Mariella Dono, Hansjuerg Alder, Florencia Bullrich,
Massimo Negrini, and Carlo M. Croce
Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA 19107; Compugen USA Inc., 7 Center Drive, Suite 9, Jamesburg, NJ 08831; Department of Experimental and Diagnostic Medicine and Interdepartment Center for Cancer Research, University of Ferrara, Diagnostics of Lymphoproliferative Diseases, National Institute of Cancer, Genoa 16123, Italy; and Laboratorio di Analisi Cliniche, Ospedale Civile di la Spezia, La Spezia 19126, Italy

MicroRNAs (miRNAs) are a class of small noncoding RNA genes recently found to be abnormally expressed in several types of cancer. Here, we describe a recently developed methodology for miRNA gene expression profiling based on the development of a microchip containing oligonucleotides corresponding to 245 miRNAs from human and mouse genomes. We used these microarrays to obtain highly reproducible results that revealed tissuespecific miRNA expression signatures, data that were confirmed by assessment of expression by Northern blots, real-time RT-PCR, and literature search. The microchip oligolibrary can be expanded to include an increasing number of miRNAs discovered in various species and is useful for the analysis of normal and disease states.

Microarray analysis shows that some microRNAs downregulate large numbers of target mRNAs.
Lee P. Lim1, Nelson C. Lau2, Philip Garrett-Engele1, Andrew Grimson2,
Janell M. Schelter1, John Castle1, David P. Bartel2, Peter S. Linsley1
& Jason M. Johnson1
1Rosetta Inpharmatics (wholly owned subsidiary of Merck and Co.), 401 Terry
Avenue N, Seattle, Washington 98109, USA
2Whitehead Institute for Biomedical Research and Department of Biology,
Massachusetts Institute of Technology, 9 Cambridge Center, Cambridge, Massachusetts 02142, USA

MicroRNAs (miRNAs) are a class of noncoding RNAs that post-transcriptionally regulate gene expression in plants and animals1,2. To investigate the influence of miRNAs on transcript levels, we transfected miRNAs into human cells and used microarrays to examine changes in the messenger RNA profile.Here we show that delivering miR-124 causes the expression profile to shift towards that of brain, the organ in which miR-124 is preferentially expressed, whereas delivering miR-1 shifts the profile towards that of muscle, where miR-1 is preferentially expressed. In each case, about 100 messages were downregulated after 12 h. The 30 untranslated regions of these messages had a significant propensity to pair to the 50 region of the miRNA, as expected if many of these messages are the direct targets of the miRNAs3. Our results suggest that metazoan miRNAs can reduce the levels of many of their target transcripts, not just the amount of protein deriving from these transcripts. Moreover, miR-1 and miR-124, and presumably other tissue-specific miRNAs, seem to downregulate a far greater number of targets than previously appreciated, thereby helping to define tissue-specific gene expression in humans.

MicroRNA expression profiling using LNA-modified probes in a liquid-phase bead-based array.

Here we describe a new liquid-phase, bead-based array for in-solution expression analysis of microRNAs (miRNAs). The array combines Locked Nucleic Acid (LNA™)-modified capture probes and the xMAP® multiplexing bead technology. Incorporation of LNA in the array capture probes greatly increases their affinity for their short miRNA targets, thereby adding selectivity to the array. As a result, the FlexmiR™ system offers a highly specific, robust and fast miRNA profiling platform.

miRCURY™ LNA research tools for microRNA.

As a relatively new but fast-growing area for research, microRNAs (miRNAs) present new challenges for researchers, based primarily on the small size of their target. The intrinsic properties of the nucleotide analog locked nucleic acid (LNA™) are used by Exiqon as the basis for a range of enabling tools for studying miRNA: the miRCURY LNA product line. The miRCURY LNA products include tools for miRNA profiling on arrays, miRNA detection—by in situ hybridization and northern blotting, and for studying miRNA function by specific knockdown of miRNAs.